Method of isolating nucleic acid having desired functional property and kit therefor

ABSTRACT

The present invention provides a method for isolating a nucleic acid having an intended functional property conveniently and rapidly, as well as a kit for carrying out the method. Specifically, the present invention provides a method comprising the steps of: (A) transferring a nucleic acid into a plurality of first host cells and allowing the nucleic acid to transiently express therein; (B) selecting, from the first host cells into which the nucleic acid is transferred, a cell which a nucleic acid having an intended functional property has been transferred; (C) preparing a purified nucleic acid from the selected cell; and (D) selecting a purified nucleic acid having an intended functional property, as well as a kit to carry out the method.

TECHNICAL FIELD

The present invention relates to a method of isolating a nucleic acidhaving an intended functional property and a kit for carrying out themethod.

BACKGROUND ART

Expression cloning is a method of cloning a gene that uses a functionexhibited as a result of expression of the target gene as an index. Thismethod does not requires information such as the base sequence of thegene or the amino acid sequence of the gene product, and is advantageouswhen cloning genes whose expression amount is small, and/or genes forwhich only functional information is available.

The gene products of mammals include many proteins that interact withother factors (such as proteins) in mammalian cells. Therefore, whenconducting expression cloning of genes from mammalian cells, it ispreferable to use the mammalian cells from which the gene is derived.

In the case where expression cloning is conducted using a mammalian cellaccording to conventional methods, even when a mammalian cell containingnucleic acid having the intended functional property is isolated,usually the isolated mammalian cell simultaneously contains plural kindsof nucleic acids in a single cell, in contrast to prokaryotes and fungi.For this reason, it is necessary to purify the nucleic acid having anintended functional property from the plurality of nucleic acidsexisting in the isolated cell.

When conventional methods are used, a large amount of labor is requiredin order to purify a nucleic acid having an intended functional propertyfrom an isolated cell. This may be attributed to the following fact;during expression screening using mammalian cells, a known retrovirus oradenovirus is used as a vector system to stably carry a foreign gene,however, these virus vector systems incorporate several vector genesinto the chromosome of a host call, thus in order to purify and identifythe transferred nucleic acid the purification process has to be repeatedseveral times. In order to obtain a purified clone, a typicalpurification process comprises the steps of:

1) PCR amplification of the nucleic acid sequence;

2) transfection of the host cell with the amplified PCR fragment; and

3) selection of transfected cells based on a desired phenotype, and theseries of the steps should be repeated 10 to 20 times

Methods of cloning and purifying a mixture of nucleic acids usingmicroorganisms such as Escherichia coli are well known. Theoretically,after isolating a first host cell containing plural kinds of nucleicacids, a specific nucleic acid may be purified from the plural kinds oftransferred nucleic acids using a second host cell such as Escherichiacoli. Such purification methods require, for example, the steps ofamplifying a nucleic acid incorporated into a chromosome by PCR or thelike; ligating the amplified nucleic acid into a vector thatautonomously replicates in a second host cell; and transferring theligated product into a second host cell. However, this ligation step isvery inefficient, and as the number of individual nucleic acids to beligated increases, the chances of succeeding in purifying the targetnucleic acid reduces. For this reason, in conventional methods usingretroviruses, adenoviruses and the like, a second host cell such asEscherichia coli is not used for the purpose of purifying a specificnucleic acid, for example, during expression cloning.

As a means for transferring a foreign nucleic acid into a mammaliancell, besides the method of using viruses such as retroviruses,conventional methods such as calcium precipitation are also well-known.However, when transient expression is conducted using conventionalmethods such as calcium precipitation, unlike methods usingretroviruses, adenoviruses or the like, degradation of the nucleic acidoccurs during transduction, and the nucleic acid fragment encoding thetransferred foreign gene is unstable in the host cell. These problemsare due to an intrinsic complication of conventionally used methods oftransient expression, and thus transient expression systems such ascalcium precipitation are thought to be unsuited as means for obtaininga clone.

Eventually, in conventional methods, even if a candidate nucleic acid isisolated, the candidate gene is usually a population of clones includinga plurality of different clones, thus it is necessary to purify andisolate a single clone from the population of clones. This purificationrequires a large amount of labor in terms of multiple screening steps,and this obstructs practical isolation and screening.

Therefore, it is an object of the present invention to provide a novelmethod of simply and rapidly isolating a nucleic acid (e.g., screeningmethod), and to provide a kit for carrying out the method.

SUMMARY OF THE INVENTION

Therefore, the present invention provides a novel method of isolating anucleic acid conveniently and rapidly (e.g., screening method), as wellas a kit for carrying out the method. By using the method and kit of thepresent invention, it is possible to carry out screening, particularlyexpression screening using mammalian cells, rapidly and conveniently.Conventionally, during expression screening using mammalian cells, alarge amount of labor and time was required in order to purify acandidate nucleic acid, however the required labor and time can largelybe reduced using the present invention. Therefore, the effect of thepresent invention is significant.

The present invention has the following features:

(1) A method of isolating a nucleic acid having an intended functionalproperty, comprising the steps of:

(A) transferring a nucleic acid into a plurality of first host cells andallowing the nucleic acid to transiently express therein;

(B) selecting, from the plurality of first host cells into which thenucleic acid is transferred, a cell into which a nucleic acid having anintended functional property has been transferred;

(C) preparing a purified nucleic acid from the selected cell; and

(D) selecting a purified nucleic acid having an intended functionalproperty.

(2) The method according to item (1), wherein at least two kinds ofnucleic acids are transferred into the plurality of first host cells.

(3) The method according to item (1), wherein the step of transferring anucleic acid into the plurality of first host cells is carried outaccording to a procedure selected from the group consisting of: atransferring method using a viral envelope, a transferring method usinga liposome, a transferring method using a liposome containing at leastone protein from a viral envelope, a transferring method using calciumphosphate and an electroporation method.

(4) The method according to item (1), wherein the nucleic acid includesa foreign gene and a promoter.

(5) The method according to item (1), wherein the host cells aremammalian cells.

(6) The method according to item (1), wherein the host cells are humancells.

(7) The method according to item (1), wherein the viral envelope isderived from wild-type or recombinant viruses.

(8) The method according to item (1), wherein the viral envelope isderived from a virus belonging to a family selected from the groupconsisting of Retroviridae, Togaviridae, Coronaviridae, Flaviviridae,Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae,Poxyiridae, Herpesviridae, Baculoviridae and Hepadnaviridae.

(9) The method according to item (8), wherein the virus is derived fromviruses belonging to the family Paramyxoviridae.

(10) The method according to item (9), wherein the virus is HVJ.

(11) The method according to item (1), wherein the vector is a viralenvelope vector.

(12) The method according to item (1), wherein the vector is a vectorcontaining a protein prepared from a viral envelope and a liposome.

(13) The method according to item (12), wherein the protein preparedfrom a viral envelope is a protein selected from the group consisting ofF protein, HN protein, NP protein and a combination thereof.

(14) The method according to item (1), wherein the step (C) of preparinga purified nucleic acid from the selected cell is carried out in thefollowing steps of:

(i) extracting a nucleic acid from the selected cell;

(ii) transferring the extracted nucleic acid into a second host cell tothereby obtain a transformed cell;

(iii) purifying the transformed cell; and

(iv) preparing a nucleic acid from the purified transformed cell.

(15) The method according to item (14), wherein the second host cell isa bacterium or a fungus.

(16) The method according to item (15), wherein the nucleic acidcontains a sequence that is necessary for autonomous replication in thebacterium or fungus.

(17) The method according to item (15), wherein the bacterium belongs toa genus selected from the group consisting of Escherichia, Bacillus,Streptococcus, Staphylococcus, Haemophilus, Neisseria, Actinobacillusand Acinetobacter.

(18) The method according to item (17), wherein the bacterium isEscherichia coli.

(19) The method according to item (15), wherein the fungus isSaccharomyces, Schizosaccharomyces or Neurospora.

(20) The method according to item (1), wherein the step (D) of selectinga purified nucleic acid having an intended functional property iscarried out in the following steps of:

(i) transferring the purified nucleic acid into a third host cell toobtain a transformed cell;

(ii) comparing the property of the transformed cell with the property ofa third host cell that is not transformed; and

(iii) determining whether or not the transformed cell has an intendedfunctional property, as a result of the comparison.

(21) The method according to item (20), wherein the step (D) ofselecting a purified nucleic acid having an intended functional propertyfurther includes the step of (iv) preparing a nucleic acid having anintended functional property from the selected cell.

(22) The method according to item (20), wherein the third host cell is amammalian cell.

(23) The method according to item (20), wherein the third host cell is ahuman cell.

(24) The method according to item (20), wherein the third host cell isderived from the same species as the species from which the first hostcell is derived.

(25) The method according to item (1), wherein the intended functionalproperty is selected from the group consisting of induction ofangiogenesis, tumor suppression, enhancement of osteogenesis, inductionof apoptosis, cytokine secretion, induction of dendrites, suppression ofarteriosclerosis, suppression of diabetes; suppression of autoimmunediseases; suppression of Alzheimer's disease, suppression of Parkinson'sdisease, protection of nerve cells and combinations thereof.

(26) A kit for isolating a nucleic acid having an intended functionalproperty, comprising:

(A) a nucleic acid transfer vector to be transferred into a plurality ofthe first host cells in order to transform said first host cells; and

(B) a second host cell for preparing a purified nucleic acid from a cellselected from the transformed first host cells.

(27) The kit according to item (26), wherein the nucleic acid transfervector is a viral envelope, liposome or liposome containing at least oneprotein from viral envelope.

(28) The kit according to item (26), wherein the first host cells aremammalian cells.

(29) The kit according to item (26), wherein the first host cells arehuman cells.

(30) The kit according to item (26), wherein the viral envelope isderived from wild-type or recombinant viruses.

(31) The kit according to item (26), wherein the viral envelope isderived from a virus belonging to a family selected from the groupconsisting of Retroviridae, Togaviridae, Coronaviridae, Flaviviridae,Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae,Poxyiridae, Herpesviridae, Baculoviridae and Hepadnaviridae.

(32) The kit according to item (26), wherein the virus is derived fromviruses belonging to the family Paramyxoviridae.

(33) The kit according to item (26), wherein the virus is HVJ.

(34) The kit according to item (26), wherein the vector is a viralenvelope vector.

(35) The kit according to item (26), wherein the vector is a vectorcontaining a protein prepared from a viral envelope and a liposome.

(36) The kit according to item (35), wherein the protein prepared from aviral envelope is a protein selected from the group consisting of Fprotein, HN protein, NP protein and a combination thereof.

(37) The kit according to item (26), wherein the second host cell is abacterium or fungus.

(38) The kit according to item (26), further comprising a nucleic acidfor preparing a nucleic acid to be transferred into the first hostcells.

(39) The kit according to item (26), further comprising a reagent to beused for determining whether or not the purified nucleic acid has anintended functional property.

(40) The kit according to item (37), wherein the bacterium belongs to agenus selected from the group consisting of Escherichia, Bacillus,Streptococcus, Staphylococcus, Haemophilus, Neisseria, Actinobacillusand Acinetobacter.

(41) The kit according to item (40), wherein the bacterium isEscherichia coli.

(42) The kit according to item (37), wherein the fungus isSaccharomyces, Schizosaccharomyces or Neurospora.

(43) A nucleic acid isolated by the method according to item (1).

(44) Use of a viral envelope for isolating a nucleic acid having anintended functional property.

(45) Use of a liposome containing at least one protein from a viralenvelope, for isolating a nucleic acid having an intended functionalproperty.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the steps in genome screening using HVJ-E.

FIG. 2 shows the result of an HAEC cell growth assay after transferringa genomic library gene.

FIG. 3 is a computer-generated graph, showing the cell growth states ofeach well in a human heart pcDNA library screening.

FIG. 4 is a schematic showing confirmation of an insert by digestingcloned genes using restriction enzymes.

FIG. 5 shows the result of a second cell growth assay.

FIG. 6 shows micrographs of each well at 40-fold magnification.

FIG. 7 is a graph comparing areas of cells exhibiting angiogenesis(left) and a graph comparing the length of cells exhibiting angiogenesis(right) obtained by using an angiogenesis quantification software.

FIG. 8 is a graph comparing joint numbers of the junctions of cellsexhibiting angiogenesis (left) and a graph comparing the numbers ofpaths of cells exhibiting angiogenesis (right).

FIG. 9 is a graph comparing the effect of the clone on c-fos genepromoter activity.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the following specification explaining the present invention,it is to be understood that articles for singular forms (e.g., “a”,“an”, “the” in English, “ein”, “der”, “das”, “die” and their declinedforms in German, “un”, “une”, “le”, “la” in French, and “un”, “una”,“el”, “la” in Spanish, and corresponding articles and adjectives inother languages) also imply concepts of plural forms, unless otherwiseindicated. In addition, the terms used in this specification should beunderstood as being used in the sense that is generally used in the art,unless otherwise indicated.

(Definition)

The term “cell” as used herein is defined in a similar manner to thebroadest meaning used in the art, and refers to a living organism whichis a sub-unit of tissue of a multicellular organism, encapsulated by amembrane structure that separates it from the external environment, isself-reproducing and carries genetic information and an expressionsystem therefore.

The terms “protein”, “polypeptide” and “peptide”, as used herein may beinterchangeably used, and each term refers to a macromolecule (polymer)comprising a sequence of amino acids. The term “amino acid” refers to anorganic molecule having a carboxylic group and an amino group at acarbon atom. Preferably, the amino acids used in this specification areusually, but are not limited to, the 20 naturally occurring amino acids.

The terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and“oligonucleotide” as used herein are interchangeably used unlessotherwise indicated, and each term refers to a macromolecule (polymer)comprising a sequence of nucleotides. The term “nucleotide” refers to anucleoside in which the 5′ moiety of ribose is a phosphate ester.Nucleotides having a pyrimidine base or purine base (pyrimidinenucleotide and purine nucleotide) as a base moiety are known. Apolynucleotide includes deoxyribonucleic acid (DNA) or ribonucleic acid(RNA).

This term also includes “derivative oligonucleotide” or “derivativepolynucleotide”. The term “derivative oligonucleotide” or “derivativepolynucleotide” is interchangeably used, and refers to anoligonucleotide or polynucleotide which contains a derivative of anucleotide or an oligonucleotide in which the bond between two or morenucleotides is not the normal one.

Concrete examples of such oligonucleotides include:2′-O-methyl-ribonucleotide; a derivative oligonucleotide in which aphosphodiester bond in the oligonucleotide is changed to aphosphorothioate bond; a derivative oligonucleotide in which aphosphodiester bond in the oligonucleotide is changed to a N3′-P5′phosphoroamidate bond; a derivative oligonucleotide in which a riboseand phosphodiester bond in the oligonucleotide is changed into apeptide-nucleic acid bond; a derivative oligonucleotide in which auracil in the oligonucleotide is substituted by C-5 propynyluracil; aderivative oligonucleotide in which a uracil in the oligonucleotide issubstituted by C-5 thiazole uracil; a derivative oligonucleotide inwhich a cytosine in the oligonucleotide is substituted by C-5 propynylcytosine; a derivative oligonucleotide in which a cytosine in theoligonucleotide is substituted by phenoxazine-modified cytosine; aderivative oligonucleotide in which a ribose moiety in a DNA molecule issubstituted by 2′-O— propyl ribose; and a derivative oligonucleotide inwhich a ribose moiety in the oligonucleotide is substituted by2′-methoxyethoxy ribose. Unless otherwise specified, a specific nucleicacid sequence is intended to encompass conservative variants (forexample, degenerate codon substitutes) or complementary sequencesthereof as well as the specific sequence given. Specifically, adegenerate codon substitute can be achieved by creating a sequence inwhich the third position of one or more selected (or all) codon(s) issubstituted by a mixed base and/or a deoxyinosine residue (Batzer etal., Nucleic Acid Res. 19:5081 (1991); Otsuka et al., J. Biol. Chem.260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98(1994)). The term “nucleic acid” as used herein is also usedinterchangeably with gene, cDNA, RNA, mRNA, oligonucleotide andpolynucleotide.

The term “gene” as used herein means a factor that defines inheritedcharacteristics. Usually, genes are arranged on a chromosome in acertain order. A gene that defines the primary structure of protein iscalled a structural gene, and a gene that regulates the expression of astructural gene is called a regulatory gene. In this specification, theterm “gene” sometimes refers to a “polynucleotide”, “oligonucleotide”and “nucleic acid”.

The term “fragment” of a nucleic acid molecule as used herein refers toa polynucleotide that is shorter than the entire length of the referencenucleic acid molecule, but has a length sufficient for use as a factorof the present invention. Therefore, a fragment as used herein is apolypeptide or a polynucleotide having a sequence length of 1 to n-1relative to the full length of a polypeptide or a polynucleotide (havinga length of n) The length of the fragment may be appropriately selecteddepending on the purpose thereof, and a lower limit of length for apolypeptide can include 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50and more amino acids. Lengths represented by integers not specificallyrecited above (for example, 11) are also suitable as a lower limit.Polynucleotides can include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,75, 100 and more nucleotides. Lengths represented by integers notspecifically recited above (for example, 11) are also suitable as alower limit.

The term “homology” of a gene as used herein represents the degree ofidentity to each other between two ore more gene sequences. Therefore,the higher the homology of two specific genes, the higher the identityand similarity of their sequences are. Whether or not two selected geneshave homology may be examined by a direct comparison of their sequences,or hybridization under stringent conditions in the case of nucleic acidsequences. During direct comparison of two gene sequences, typically,when at least 50%, preferably at least 70%, more preferably, when atleast 80%, 90%, 95%, 96%, 97%, 98% or 99% of the DNA sequences areidentical between the two gene sequences, the genes are determined tohave homology.

Comparison of similarity and identity between base sequences anddetermination of homology of base sequences are carried out herein bymeans of BLAST which is a sequence analysis tool using defaultparameters.

“Expression” of a gene, polynucleotide, polypeptide or the like refersto the phenomenon that the gene or the like takes a different form undercertain circumstances in vivo. Preferably, it means the phenomena of agene, polynucleotide or the like being transcribed and translated into apolypeptide. The phenomena of mRNA being produced as a result oftranscription may also be one form of expression. More preferably, theresultant polypeptide may have undergone post-translational processing.

The term “a polynucleotide that hybridizes under stringent conditions”as used herein refers to well-known conditions that are commonly used inthe art. Such a polynucleotide can be obtained by the colonyhybridization method, the plaque hybridization method or Southern blothybridization using a polynucleotide selected from the polynucleotidesof the present invention as a probe. Specifically, it means apolynucleotide that can be identified in the following manner. A filteron which DNA derived from colonies or plaques is immobilized is used tocarry out a polynucleotide hybridization in the presence of 0.7-1.0 MNaCl at 65° C. Then, the filter is washed at 65° C. with ×0.1 to ×2concentration of SSC (saline-sodium citrate) solution (150 mM sodiumchloride, 15 mM sodium citrate). Hybridization may be conductedaccording to methods described in laboratory manuals such as MolecularCloning 2nd ed., Current Protocols in Molecular Biology, Supplement1-38, DNA Cloning 1: Core Techniques, A Practical Approach, SecondEdition, Oxford University Press (1995) and the like. Herein, “asequence that hybridizes under stringent conditions” preferably excludessequences comprising exclusively A or exclusively T.

The wording “hybridizable polynucleotide” refers to a polynucleotidethat is able to hybridize with other polynucleotides under theaforementioned hybridization conditions. Specific examples ofhybridizable polynucleotides include polynucleotides having at least 60%or higher homology, preferably 80% or higher homology, more preferably95% or higher homology with a DNA base sequence encoding a polypeptidehaving the amino acid sequence set forth in the SEQ ID NO:2, 4 or 6. Asto the given homology, similarity may be represented by a score, forexample, by using the search program BLAST using the algorithm developedby Altschul et al. (J. Mol. Biol. 215, 403-410 (1990)).

Amino acids may be denoted herein by the generally known three-lettercoding or the one-letter coding recommended by IUPAC-IUB BiochemicalNomenclature Commission. Likewise, nucleotides may be denoted by thecommonly accepted one-letter coding.

“Corresponding” amino acid used herein refers to an amino acids havingor expected to have a similar effect in a protein or polypeptide to thespecific amino acid in a protein or polypeptide which is the referencefor comparison. In an enzyme molecule, in particular, it means an aminoacid which is located at a similar position in an active site andsimilarly contributes to catalytic activity.

The term “nucleotide” as used herein may be naturally occurring or maynot be naturally occurring. The term “derivative nucleotide” or“nucleotide analogue” refers to a nucleotide that is different from anaturally occurring nucleotide, but has a function similar to that ofthe function of the original nucleotide. Such derivative nucleotides andnucleotide analogues are well known in the art. Examples of suchderivative nucleotides and nucleotide analogues include, but are notlimited to, phosphorothioate, phosphoroamidate, methyl phosphonate,chiral methyl phosphonate, 2-O-methylribonucelotide and peptide-nucleicacid (PNA).

The term “fragment” as used herein refers to a polypeptide orpolynucleotide having a sequence length of 1 to n-1 relative to the fulllength of a polypeptide or polynucleotide (having a length of n). Thelength of the fragment may be appropriately selected depending on thepurpose thereof, and a lower limit of length for a polypeptide includes3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids.Lengths represented by integers not specifically recited above (forexample, 11) are also suitable as a lower limit. For polynucleotides,fragment includes polynucleotides of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,40, 50, 75, 100 and more in length. Lengths represented by integers notspecifically recited above (for example, 11) are also suitable as alower limit.

The term “isolation (isolated)” as used herein refers to the state of acertain substance or nucleic acid, and that the substance or nucleicacid is in a state that is different from its naturally-occurring state,and that the substance or nucleic acid is not accompanied by at leastone substance or nucleic acid that accompanies it in itsnaturally-occurring state. The term to “concentrate” a nucleic acid asused herein refers to increasing the abundance of a specific nucleicacid compared to its naturally-occurring abundance. Therefore, in apreferred condition, a concentrated nucleic acid or nucleic acidcomposition contains only a specific nucleic acid.

The term to “purify” a specific substance as used herein refers tomaking the substance in it's abundantly existing state, and reducing theconcentration of substances other than the specified substance to such adegree that they will not influence the function of the specifiedsubstance. Therefore, in a preferred condition, a purified substance orsubstance-containing composition contains only the specific substance.

In this specification the terms “purification (purified)” and “cloning(cloned)” are interchangeably used. The terms “purification (purified)”and “cloning (cloned)” refer to a state of a certain substance ornucleic acid, and refer to making the abundance of the nucleic acidhigher, preferably to the level that the substance or nucleic acid isnot substantially accompanied by other kinds of substances or nucleicacids. When used in the contexts of “purification” and “cloning” herein,the term “state where substantially no other kinds of substances ornucleic acids accompany” refers to the state where these other kinds ofsubstances or nucleic acids are completely absent, or will not exert anyinfluence on the substance or nucleic acid of interest if present.Therefore, in a more preferred condition, a purified nucleic acid ornucleic acid composition contains only a specific nucleic acid.

The term “purify” a specific substance as used herein refers to makingthe substance in an abundantly existing state, and reducing theconcentration of substances other than the specified substance to such adegree that they will not influence the function of the specifiedsubstance.

The term. “gene transfer” as used herein refers to transferring adesired naturally-occurring, synthetic or recombinant gene or genefragment into a target cell in vivo or in vitro in such a manner thatthe transferred gene maintains its function. A gene or gene fragmenttransferred in the present invention encompasses DNA or RNA having aspecific sequence, or a nucleic acid which is a synthetic analoguethereof. The terms “gene transfer”, “transfection” and “transfect” asused herein are interchangeably used.

The terms “gene transfer vector” and “gene vector” as used herein areinterchangeably used. The terms “gene transfer vector” and “gene vector”refer to vectors capable of transferring a polynucleotide sequence ofinterest into a target cell. Examples of a “gene transfer vector” and“gene vector” include, but are not limited to, a “viral envelope vector”and a “liposome vector”.

The term “viral envelope vector” as used herein refers to a vector inwhich a foreign gene is encapsulated in a viral envelope or a vector inwhich a foreign gene is encapsulated in a component containing a proteinderived from a viral envelope. The virus used for preparing a genetransfer vector may be a wild-type virus or a recombinant virus.

In the present invention, examples of the virus used for preparing aviral envelope or a protein derived from a viral envelope include, butare not limited to, viruses belonging to families selected from thegroup consisting of Retroviridae, Togaviridae, Coronaviridae,Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae,Rhabdoviridae, Poxyiridae, Herpesviridae, Baculoviridae andHepadnaviridae. Preferably viruses belonging to the familyParamyxoviridae, and more preferably HVJ (Hemagglutinating Virus ofJapan, Sendai Virus) are used.

Examples of proteins derived from a viral envelope include, but are notlimited to the F protein, HN protein, NP protein and M protein of HVJ.

The term “liposome vector” as used herein refers to a vector wherein aforeign gene is encapsulated in a liposome. Examples of lipids used forpreparing a liposome vector include, but are not limited to, neutralphospholipids such as DOPE (dioleoyl phosphatidyl ethanolamine) andphosphatidyl choline; negatively charged phospholipids such ascholesterol, phosphatidyl serine and phosphatidic acid; and positivelycharged lipids such as DC-cholesterol (dimethylaminoethanecarbamoylcholesterol) and DOTAP (dioleoyl trimethylammonium propane).

The term “liposome” used herein is one type of lipid bilayer. Forexample, when a phospholipld such as lecithin is suspended at 50% (byweight) or more in water at a temperature higher than the gel-liquidcrystal phase transition temperature which is specific for saidphospholipid, a closed vesicle composed of a lipid bilayer membraneencapsulating a water phase is formed. This vesicle is called aliposome. Liposomes are generally classified as multilayered liposomes(MLV: multilamellar vesicle), in which a plurality of bilayer membranesoverlap like an onion, and unilamellar liposomes having only onemembrane. The latter may also be prepared by making a suspension ofphospholipids such as phosphatidyl choline dispersed by intensivestirring with a mixer, followed by an ultrasound treatment.

Liposomes having only one membrane are further classified into smallsingle membrane liposomes (SUV: small unilamellar vesicle) and largesingle membrane liposomes (LUV: large unilamellar vesicle) according totheir diameter. MLVs are prepared by adding water to a lipid thin filmand applying mechanical oscillation. SUVs may be prepared byultrasonication of MLVs or by removal of a surfactant from a mixture oflipid and surfactant by dialysis or the like. Besides the above methods,other well-known methods include (1) a method of preparing LUV bytreating SUV with multiple freeze-thaw cycles; (2) a method of preparingLUVs by fusing SUVs composed of acidic phospholipids in the presence ofCa²⁺ and then removing the Ca²⁺ with EDTA (ethylenediamine tetraaceticacid), and (3) a method of preparing LUVs and the like by causing phaseconversion while distilling off ether from an emulsion of lipids insolution with ether and water (reverse-phase evaporation vesicle: REV).

The terms “surfactant” and “surface activator” as used herein areinterchangeably used. A surfactant is a substance that exhibits strongsurface-tension activity against water, and forms an aggregate such asmicelle in a solution at concentrations exceeding the critical micelleconcentration. A surfactant has both a hydrophilic moiety and ahydrophobic (lipophilic) moiety, and strongly absorbs into a two-phaseinterface of water and oil according to the balance of hydrophilicityand lipophilicity, to significantly decrease the free energy (interfacetension) at the interface. Typical hydrophobic groups are long chainhydrocarbon groups such as alkyl groups; typical hydrophilic groups mayinclude ionic dissociation groups and nonionic polar groups such as ahydroxyl group. Since surfactants having a carboxyl group, sulfo group,hydrogen sulfate group, and —OSO—OH dissociate in water to becomeanions, they may be called anionic surfactants. Typical examplesinclude, but are not limited to, fatty acid soaps, alkyl benzenesulfonate and the like. In contrast, those having a quaternary ammoniumgroup dissociate to become cations and are called cationic surfactants.There are also surfactants having both a cationic dissociation group andan anionic dissociation group in the same molecule, such as long chainalkyl amino acids, and such surfactants are called amphotericsurfactants. Those surfactants having a nonionic polar group are callednonionic surfactants, polyoxyethylenenonylphenyl ether is typicalexample of a non-ionic surfactant.

The term “lipid” as used herein encompasses any lipids, as long as (1)they have a long chain fatty acid or a similar hydrocarbon chain in themolecule, and (2) they exist in an organism or they are moleculesderived from an organism. Preferred lipids are phospholipids capable offorming liposomes, and more preferred lipids include, but are notlimited to, phosphatidyl choline, phosphatidyl serine, phosphatidylinositol, phosphatidyl ethanol amine, cholesterol, sphingomyelin andphosphatidic acid.

The term “fatty acid” as used herein refers to aliphatic monocarboxylicacids and aliphatic dicarboxylic acids that are obtained by hydrolysisof naturally-occurring lipids. Typical fatty acids include, but are notlimited to, arachidonic acid, palmitic acid, oleic acid and stearicacid.

The term “gene transfer activity” as used herein refers to the activityof “gene transfer” by a vector, and may be detected using a function ofthe transferred gene as an index indicator (for example, expression ofthe encoded protein and/or activity of the protein, in the case of anexpression vector).

The term “inactivation” as used herein refers to a virus with aninactivated genome. Inactivated viruses are replication defective.Preferably, the inactivation is achieved by UV treatment or treatmentwith an alkylation agent.

The term “foreign gene” as used herein refers to a nucleic acidcontained in a viral envelope vector but not originating from the virus,or a nucleic acid contained in a liposome vector. In one aspect of thepresent invention, the foreign gene is operatively linked with aregulatory sequence which allows the gene transferred by a gene transfervector to be expressed (e.g., a promoter, enhancer, terminator and apoly A addition signal are required for transcription, and a ribosomebinding site, initiation codon and a termination codon are required fortranslation). In another aspect of the present invention, the foreigngene does not include a regulatory sequence for expression of theforeign gene. In a further aspect of the present invention, the foreigngene is an oligonucleotide or a decoy nucleic acid.

A foreign gene contained in a gene transfer vector is typically anucleic acid of DNA or RNA, and the transferred nucleic acid moleculemay include a nucleic acid analogue molecule. Molecular speciescontained in a gene transfer vector may be a single gene moleculespecies or a plurality of different gene molecule species.

The term “gene library” as used herein means a nucleic acid libraryincluding nucleic acid sequences isolated from the natural world orsynthetic nucleic acid sequences. Examples of the source of nucleic acidsequences isolated from the natural world, include, but are not limitedto, genome sequences and cDNA sequences derived from eukaryotic cells,prokaryotic cells, or viruses. A library of sequences isolated from thenatural world to which optional sequences (e.g., signal sequences or tagsequences) are added is also encompassed in the gene library of thepresent invention. In one embodiment, a gene library also includessequences such as promoter sequences that enable transcription and/ortranslation of the nucleic acid sequences contained in the library.

The terms “HVJ” and “Sendai Virus” as used herein are usedinterchangeably. For example, the terms “envelope of HVJ” and “envelopeof Sendai Virus” are synonymously used herein. “Sendai Virus” as usedherein belongs to the genus paramyxovirus in the family Paramyxoviridae,and has cell fusion activity. The viral particles are enveloped, and arepolymorphic in that the particle diameter varies from 150 to 300 nm. Thegenome is a (−) strand RNA molecule having a length of about 15500bases. The virus has an RNA polymerase, is thermally unstable,hemagglutinates almost all types of erythrocyte and exhibits hemolyticactivity.

The term “HAU” used herein refers to a measure of virus activity that isable to hemagglutinate 0.5% of chicken erythrocytes, wherein 1 HAUcorresponds to about 24 million viral particles (Okada, Y. et al., BikenJournal 4, 209-213, 1961).

The term “candidate nucleic acid” as used herein may be any nucleic acidinsofar as it is an object to be purified. Herein, a population ofcandidate nucleic acids may be obtained directly from cells, a firsthost cell such as a mammalian cell as a source, or obtained as anisolated nucleic acid preparation. The population of candidate nucleicacids thus obtained is purified using a second host cell.

Examples of animal cells that may be used as a host cell include, butare not limited to, mouse myeloma cell lines, rat myeloma cell lines,mouse hybridoma cells, CHO cells which are derived from the Chinesehamster, BHK cells, African green monkey kidney cell lines, humanleucocyte-derived cell lines, the cell line HBT5637 (Japanese Laid-OpenPublication No. 63-299) and human colon cancer cell lines. Mouse myelomacell lines include, ps20, NSO and the like. Rat myeloma cell linesinclude YB2/0 or the like. Human embryo kidney cell lines include HEK293(ATCC: CRL-1573) or the like. Human leucocyte-dervied cell lines includeBALL-1 or the like. African green monkey kidney cell lines includeCOS-1, COS-7 or the like. Human colon cancer cell lines include HCT-15or the like.

The term “animal” as used herein is used in the broadest sense withinthe art and includes vertebrates and invertebrates. Examples of animalsinclude, but are not limited to, the classes Mammalia, Aves, Reptilia,Amphibia, Pisces, Insecta, Vermes and the like.

The term “tissue” of an organism as used herein refers to a populationof cells having a certain similar ability across the population.Therefore, the tissue may be a part of an organ. A particular organoften has cells having the same function, however, it may include cellshaving slightly different functions. Therefore, in this specification, avariety of cells may be included in a particular tissue insofar as theycommonly have a certain characteristic.

The second host cell is not particularly limited insofar as it is a cellcapable of “gene-transferring” a candidate nucleic acid; various hostcells that are conventionally used in genetic engineering (for example,prokaryotic cells and eukaryotic cells) may be used.

Examples of prokaryotic cells include, prokaryotic cells belonging tothe genus selected from the group consisting of Escherichia, Bacillus,Streptococcus, Staphylococcus, Haemophilus, Neisseria, Actinobacillus,Acinetobacter, Serratia, Brevibacterium, Corynetbacterium,Microbacterium, and Pseudomonas, for example, Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coliMC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichiacoli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichiacoli W3110, Escherichia coli NY49, Escherichia coli BL21 (DE3),Escherichia coli BL21 (DE3) pLysS, Escherichia coli HMS174 (DE3),Escherichia coli HMS174 (DE3) pLysS, Serratia ficaria, Serratiafonticola, Serratia liquefaciens, Serratia marcescens, Bacillussubtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes,Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticumATCC14066, Corynebacterium glutamicum ATCC13032, Corynebacteriumglutamicum ATCC14067, Corynebacterium glutamicum ATCC13869,Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilumATCC15354, Pseudomonas sp D-0110 and the like.

Examples of eukaryotic cells include, yeast strains belonging to thegenus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon,Schwanniomyces, Pichia, and fungi belonging to Neurospora, specifically,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastorisand the like. As a method of transferring a recombinant vector in thehost cells, any method for transferring DNA into fungi can be used, suchmethods include electroporation methods [Methods Enzymol., 194, 182(1990)], spheroplast-based methods [Proc. Natl. Acad. Sci. USA, 84, 1929(1978)], lithium acetate-based methods [J. Bacteriol., 153, 163 (1983)]and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

Plant cells, for example, include plant cells derived from potato,tobacco, corn, rice plant, rapeseed, soy bean, tomato, carrot, wheat,barley, rye, alfalfa and flax. As a method of transferring a recombinantvector, any method for transferring DNA into a plant cell can be used.Examples of such methods include the use of Agrobacterium (JapaneseLaid-Open Publication No. 59-140885, Japanese Laid-Open Publication No.60-70080, WO94/00977), electroporation methods (Japanese Laid-OpenPublication No. 60-251887) and methods using a particle gun (gene gun)(Japanese patent No. 2606856, Japanese patent No. 2517813).

Insect cells may include Spodoptera frugiperda ovary cells,Trichopllusia ni ovary cells, cultured cells derived from silkwormovaries and the like. Examples of Spodoptera frugiperda ovary cellsinclude Sf9, Sf21 (Baculovirus Expression Vectors: A Laboratory Manual)and the like, examples of Trichopllusia ni ovary cells include High 5,BTI-TN-5B 1-4 (Invitrogen) and the like, and examples of the culturecells derived from silkworm ovaries include Bombyx mori N4.

The term “variant” as used herein refers to substances which arepartially modified from the original substances such as polypeptides orpolynucleotides. A variant includes substitution variants, additionvariants, deletion variants, truncated variants, allelic mutants and thelike. Alleles are genetic variants which belong to the same locus butare distinct from each other. Therefore, the term “allelic gene mutant”means a variant which forms an allelic relative to a certain gene. Theterm “species homolog or homolog” denotes to that the substance has ahomology (preferably, homology of 60% or more, more preferably, homologyof 80% or more, 85% or more, 90% or more, 95% or more) with a certaingene in a certain species at the amino acid level or nucleotide level. Amethod for obtaining such a species homolog is apparent from thedescription of this specification. An “ortholog” is also called an“orthologous gene”, the term is used for two genes that result fromspeciation of a specific common ancestor. Taking the hemoglobin genefamily having a multigene structure as an example, human and mouseα-hemoglobin genes are orthologs, but the human α-hemoglobin andβ-hemoglobin genes are paralogs (genes generated as a result of geneduplication). Since orthologs are useful for estimating a molecularphylogenetic tree, orthologs may also be useful in the presentinvention.

The term “conservative (conservatively modified) variant” is applicableboth to amino acid sequences and nucleic acid sequences. Regarding aspecific nucleic acid sequence, a conservative variant refers to anucleic acid that encodes the same or substantially the same amino acidsequence. When the nucleic acid does not encode an amino acid sequence,it refers to substantially the same sequence. Because of the degeneracyof genetic codes, a large number of functionally equivalent nucleicacids encode any particular protein. For example, the codons GCA, GCC,GCG and GCU all encode the amino acid, alanine. Therefore, at everyposition where a codon specifies alanine, the codon may be changed intoany corresponding codon described above without changing the encodedpolypeptide. Such variation of nucleic acids is “silent variant(mutation)” which is one of the conservative variants. Any nucleic acidsequence encoding a polypeptide herein also describes all possiblesilent mutations for the nucleic acid. One skilled in the art willrecognize that each codon in a nucleic acid (excluding AUG which isusually a unique codon for methionine, and TGG which is usually a uniquecodon for tryptophan) may be modified so as to produce a functionallyidentical molecule. Therefore, all possible silent mutations of anucleic acid encoding a polypeptide is implied in each of the describedsequences. Preferably, such modification may be made so as to avoidsubstitution of cysteine which is an amino acid that exerts greatinfluence on the higher structure of polypeptides.

In order to create a functionally equivalent polypeptide, addition,deletion or modification of amino acid may be carried out herein besidessubstitution of amino acid. Substitution of an amino acid refers tomaking a substitution in the original peptide with at least one aminoacid, for example 1 to 10, preferably 1 to 5, more preferably 1 to 3amino acids. Addition of an amino acid refers to adding onto theoriginal peptide, at least one amino acid, for example 1 to 10,preferably 1 to 5, more preferably 1 to 3 amino acids. Deletion of anamino acid refers to deleting from the original peptide, at least oneamino acid, for example 1 to 10, preferablyl to 5, more preferably 1 to3 amino acids. Examples of amino acid modification include, but are notlimited to, amidation, carboxylation, sulfation, halogenation,alkylation, glycosylation, phosphorylation, hydroxylation and acylation(for example, acetylation). Amino acids that are substituted or addedmay be naturally occurring amino acids, non-naturally occurring aminoacids, or amino acid analogues. Naturally occurring amino acids arepreferred.

Such a nucleic acid may be obtained using well-known PCR methods orchemical synthesis. Site-directed mutagenesis, hybridization methods andthe like may be combined with the methods described above.

The term “substitution, addition or deletion” of a polypeptide orpolynucleotide as used herein refers to the occurrence of substitution,addition or deletion of an amino acid or it's alternative, or anucleotide or it's alternative from the original polypeptide orpolynucleotide. Techniques for substitution, addition or deletion arewell known in the art, and include, for example, site-directedmutagenesis and the like. The number of substitutions, additions ordeletions is at least one, and any number is acceptable insofar as afunction of interest (for example, a cancer marker, a neurologicaldisease marker or the like) is retained in a variant having such asubstitution, addition or deletion. For example, the number may be oneor several, preferably within 20%, within 10% of the entire length, orless than or equal to 100, less than or equal to 50, or less than orequal to 25.

General molecular biological methods that may be used herein can bereadily practiced by a person skilled in the art with reference to, forexample, Ausubel F. A. et al. ed. (1988), Current Protocols in MolecularBiology, Wiley, New York, N.Y.; Sambrook J. et al. (1987) MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

The term “expression plasmid” as used herein refers to a nucleic acidsequence in which, in addition to a structural gene and a promoter forregulating expression thereof, various regulatory elements are linked soas to be able to operate in a host cell. Preferably, the regulatoryelements may include a promoter, a terminator and a selection marker. Itis well-known by those skilled in the art that the type of expressionplasmid and the kind of regulatory element used may vary depending onthe host bacterial cell. In the present invention, an expression plasmidexpresses a candidate nucleic acid in a first host cell and/or secondhost cell. Therefore, the expression plasmid includes a candidatenucleic acid, and a regulatory element (for example, promoter) operablylinked with the candidate nucleic acid.

The term “promoter” used herein refers to a region of DNA thatdetermines the transcription initiation site of a gene and directlyregulates the frequency of transcription thereof, and a base sequencewhere RNA polymerase binds to start transcription. A putative promoterregion varies with the particular structural gene, and is usuallypositioned upstream of a structural gene. Not limited to these, aputative promoter may also be positioned downstream of a structuralgene.

A promoter may be inducible, constitutive, site specific, or periodspecific. As the promoter, any promoters that can be expressed in a hostcell such as mammalian cells, Escherichia coli, yeast or the like areacceptable.

When used herein concerning the expression of a gene, the term “sitespecificity” generally refers to the specificity of expression of thegene within specific mammalian tissues. The term “period specificity”refers specifically to expression of the gene in accordance with thedevelopmental stage of a mammal. Such specificity may be introduced intoa desired organism by selecting an appropriate promoter.

When the expression of a promoter of the present invention is describedas “constitutive” herein, it means that in all tissues in organism, thegene is expressed in an almost constant amount regardless of thegrowth/proliferation of the organism. More specifically, if duringNorthern blotting analysis almost the same level of expression isobserved both in the same or in a corresponding site at given points(for example, two or more points (for example, at 5 days, and 15 days))the expression is said to be constitutive according to the definition ofthe present invention. Constitutive promoters are believed to play arole in maintaining the homeostasis of organisms in normal growthenvironments. The term that the expression of a promoter of the presentinvention is “responsive” means that when at least one factor is givento an organism, the level of expression changes. In particular, whenexpression levels increase in response to at least one factor theexpression is said to be “inducible” by the factor, and when expressionlevels reduce in response to at least one factor the expression is saidto be “reductive” by the factor. “Reductive” expression is based on thepremise the fact that expression is initially observed under normalconditions, and hence “reductive” expression is concept that overlaps“constitutive” expression. These properties may be determined byanalyzing RNA extracted from a specific tissue of an organism in orderto analyze the expression levels by Northern blotting analysis or byquantitating the expressed protein by Western blotting. A mammalian cellor a mammal (including a specific tissue) transformed with a vector thatincorporates a promoter inducible by a factor, as well as a nucleic acidencoding a site specific recombinant inducing factor of the presentinvention, may be subjected to site specific recombination of the sitespecific recombination sequence under certain conditions by using astimulating factor having the function of inducing the promoter.

As a potent promoter for expression in a mammalian cell, a variety ofnaturally occurring promoters (for example, the early promoter of SV40,the EIA promoter of adenovirus, the promoter of human cytomegarovirus(CMV), the human elongation factor-1 (EF-1)*promoter, the promoter ofthe Drosophila minimum heat shock protein 70 (HSP), the humanmetallothionein (MT) promoter, the Rous-sarcoma virus (RSV) promoter,the human ubiquitin C (UBC) promoter, human actin promoter), andartificial promoters (for example, fusion promoters such as SRα promoter(a fusion of the SV40 early promoter and the LTR promoter of HTLV) andthe CAG promoter (a hybrid of the CMV-IE enhancer and the chicken actinpromoter)) are well known. Therefore, by using these well-knownpromoters or variants thereof, it is possible to readily increase theexpression level.

When Escherichia coli is used as a host cell, promoters derived fromEscherichia coli or phages such as the trp promoter (Ptrp), the lacpromoter (Plac), the PL promoter, the PR promoter, the PSE promoter, theSPO1 promoter, the SPO2 promoter, the penP promoter and the like can beexemplified. Also artificially designed and modified promoters such as apromoter comprising two serially linked Ptrps (Ptrp ×2), the tacpromoter, the lacT7 promoter, the let I promoter and the like may beused.

The term “enhancer” may be used herein for improving the expressionefficiency of a gene of interest. A typical enhancer when used in amammalian cell includes, but is not limited to, an enhancer of SV40. Anenhancer may be used singly or in plural, or may not be used at all.

The term “terminator” as used herein refers to a sequence that ispositioned downstream of a protein coding region of a gene, and isinvolved in the termination of transcription and the addition of a polyA tail when DNA is transcribed into mRNA. A terminator is known to beinvolved in the stability of mRNA and to influence the expression levelof a gene.

The term “operably linked” as used herein means that expression(operation) of an intended sequence is placed under the control of atranscription and translation regulatory sequence (for example, apromoter, an enhancer or the like) or under a translation regulatorysequence. In order to operably link a promoter to a gene, usually, thepromoter is located directly upstream of the gene, however, it is notnecessarily located adjacently.

As used herein, the term “biological activity” is used interchangeablywith the term “functional property”. The terms “biological activity” and“functional property” as used herein mean an activity that a certainfactor (for example, a nucleic acid) may have in an organism, andencompasses activities exerting various functions. For example, when acertain factor is a gene encoding a growth factor, the functionalproperty encompasses expressing the growth factor in a host cell, andpreferably promoting the growth of the cell by expression of the growthfactor. For example, when the certain factor is a gene encoding anenzyme, the functional property encompasses expressing the enzymaticactivity in a host cell and preferably increasing enzymatic activity toa detectable level. In another example, when the certain factor is agene encoding a ligand, the functional property encompasses expressing aligand that binds a receptor corresponding to the ligand, and preferablychanges the phenotype of a cell having the receptor corresponding to theligand, by the expression of the ligand.

In this specification, when it is necessary to transfer a nucleic acidinto a second host cell, any method for transferring DNA into a hostcell can be used. Examples of such methods include, transfection,transduction, transformation (for example, electroporation, methodsusing a particle gun (gene gun) and the like).

When referring to gene herein, the term “expression plasmid” means anucleic acid capable of expressing a gone included in a polynucleotidesequence of interest after the polynucleotide sequence of interest istransferred into a cell of interest. Expression plasmids includeplasmids having a promoter at a position suited for transcription of thepolynucleotide to be expressed.

In this specification, “detection” or “quantification” of the expressionof a nucleic acid transferred into a host cell may be achieved by usingappropriate methods including measurement of mRNA and immunologicalmeasuring methods. Examples of molecular biological measuring methodsinclude Northern blotting, Dot blotting, PCR and the like. Examples ofimmunological measuring methods include ELISA using a micro titer plate,RIA, fluorescent antibody methods, Western blotting, immunohistochemicalstaining and the like. Quantification methods include ELISA or RIA.

In this specification, “detection” or “quantification” of expression ofnucleic acids transferred into a host cell may be carried out using asolid phase (for example, a substrate, support, array, chip ormicrochip).

The terms “substrate” and “support” are used in the same meaning herein,and refer to a material (preferably solid) from which an array of thepresent invention is constructed. A material for the substrate includesany solid material having the characteristic of binding a biologicalmolecule used in the present invention via a covalent or noncovalentbond or any material that can be derivatized to have such acharacteristic.

Materials used for a substrate include any material capable of forming asolid surface, and include, but are not limited to, for example, glass,silica, silicones, ceramics, silicon dioxide, plastics, metals(including alloy), naturally occurring or synthetic polymers (forexample, polystyrene, cellulose, chitosan, dextran and nylon). Asubstrate may have a plurality of layers formed of different materials.For example, inorganic insulating materials, such as glass, quartzglass, alumina, sapphire, forsterite, silicon carbide, silicon oxide,silicon nitride and the like may be used. Also organic materials such aspolyethylene, ethylene, polypropyrene, polyisobutylene, polyethyleneterephtalate, unsaturated polyester, fluorine-containing resin,polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate,polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile,polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urearesin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer,acrylonitrile-butadiene-styrene copolymer, silicone resin, polyphenyleneoxide, polysulfone and the like may be used. In the present invention,membranes that are used for blotting such as nylon membrane,nitrocellulose membrane, PVDF membrane and the like may be used. A nylonmembrane is preferred because the result may be analyzed using aconvenient analyzing system when a nylon membrane is used. However, whenan object of high density is analyzed, the use of hard materials such asglass is preferred.

The term “chip” or “microchip” as used herein refers to an ultrasmallintegrated circuit which has a plurality of functions and forms a partof a system. As used herein, a “DNA chip” includes a substrate and DNA,and at least one DNA molecule (for example, cDNA fragment) is placed onthe substrate. As used herein, a “protein chip” includes a substrate andprotein, and at least one protein (for example, a polypeptide oroligopeptide) is placed on the substrate. As used herein, a “DNA chip”and a “protein chip” are encompassed by the terms “microchip” or simply“chip”. The term “microarray” means a chip on which at least onebiological molecule (for example, an oligonucleotide such as a cDNAfragment, or a peptide) is placed in array.

A biological molecule (for example, an oligonucleotide such as a cDNAfragment or a peptide) as used herein may be collected from an organismor may be chemically synthesized using methods known by those skilled inthe art. For example, oligonucleotides may be prepared by automatedchemical synthesis using either a DNA synthesizer or a peptidesynthesizer commercially available from Applied Biosystems or the like.Compositions and methods for automated oligonucleotide synthesis aredisclosed in, for example, U.S. Pat. No. 4,415,732, Caruthers et al.(1983) U.S. Pat. No. 4,500,707 and Caruthers (1985); U.S. Pat. No.4,668,777, Caruthers et al. (1987).

On a substrate, any number of biological molecules (for example, DNAmolecules or peptides) may be placed, and usually up to 10⁸ biologicalmolecules, and in another embodiment, up to 10⁷ biological molecules, upto 10⁶ biological molecules, up to 10⁵ biological molecules, up to 10⁴biological molecules, up to 10³ biological molecules, or up to 10²biological molecules may be placed on one substrate. In these cases, thesize of the substrate is preferably as small as possible. In particular,the size of a spot of a biological molecule (for example, a DNA moleculeor a peptide) may be as small as the size of a single biologicalmolecule (i.e., in the order of 1-2 nm). In some cases, the minimumsubstrate area is determined by the number of biological molecules onthe substrate.

The term “biological molecule” as used herein refers to moleculesassociated with organisms. As used herein, the term “organisms” refersto a biological organism including, but not limited to, animals, plants,fungi and viruses. The term “biological molecules” encompasses moleculesextracted from organisms, however, is not limited to this, and anymolecule that influences an organism is encompassed by the definition ofa biological molecule. Examples of such biological molecules include,but are not limited to, proteins, polypeptides, oligopeptides, peptides,polynucleotides, oligonucleotides, nucleotides, nucleic acids(including, for example, DNA molecules such as cDNA and genomic DNA, andRNA molecules such as mRNA, polysaccharides, oligosaccharides, lipids,small molecules (for example, hormones, ligands, signal transducers,organic small molecules and the like), and composite molecules thereof.As used herein, biological molecules may be preferably peptides, DNA orRNA.

In the case where a host cell changes due to a nucleic acid transferredinto the host cell, it is possible to “detect” or “quantify” theexpression of the nucleic acid transferred into the host cell bymeasuring the degree of the change. Examples of such changes in hostcells include, but are not limited to, changes in enzymatic activity ofa specific enzyme in a cell, changes in cell growth rate, and the like.

The term “expression amount” refers to the amount in which a polypeptideor mRNA is expressed in a cell of interest. Such an expression amountmay be the level of protein expression of the polypeptide of the presentinvention evaluated by any appropriate method, including immunologicalmeasuring methods such as ELISA, RIA, fluorescent antibodies, Westernblotting and immunohistochemical staining using a antibody of thepresent invention; or the level of mRNA expression of the polypeptide ofthe present invention evaluated by any appropriate methods, includingmolecular biological methods such as Northern blotting, dot blotting,and PCR and the like. As used herein, “expression amount” may be anabsolute value represented by a numerical unit, such as expressedweight, absorbance having correlation with expressed weight and thelike, or may be a relative value represented by a ratio relative to acontrol or comparison reference. “Change in expression amount” meansincrease or decrease in the expression amount in the protein level or inthe mRNA level of the polypeptide of the present invention as evaluatedby any appropriate method including the forgoing immunological ormolecular biological methods.

DESCRIPTION OF PREFERRED EMBODIMENTS

In the present invention, a method of isolating a nucleic acid having anintended functional property from candidate nucleic acids can be carriedout by the following steps:

(A) transferring a nucleic acid into a plurality of first host cells andallowing the nucleic acid to transiently express therein;

(B) selecting, from the plurality of first host cells into which thenucleic acid is transferred, a cell having the nucleic acid of theintended functional property transferred therein;

(C) preparing a purified nucleic acid from the selected cell; and

(D) selecting a purified nucleic acid having the intended functionalproperty.

In the above method, the candidate nucleic acids may be derived from anyorganism, and may be DNA, RNA or nucleotide analogues. The candidatenucleic acids may be one kind or a plurality of kinds.

In the above methods, the first host cells are preferably animal cells,more preferably mammalian cells including human cells, although notparticularly limited thereto. The candidate nucleic acids may betransferred into the first host cells using a variety of known methods.Typical methods include, but are not limited to, a transferring methodusing a viral envelope, a transferring method using a liposome, atransferring method using a liposome containing at least one proteinfrom a viral envelope, a transferring method using calcium phosphate,and an electroporation method. When the transferring method using aviral envelope or the transferring method using a liposome containing atleast one protein from a viral envelope is used, the virus used forpreparing the viral envelope may be derived from viruses belonging to afamily selected from the group consisting of Retroviridae, Togaviridae,Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae,Bunyaviridae, Rhabdoviridae, Poxyiridae, Herpesviridae, Baculoviridaeand Hepadnaviridae. Preferably, the virus is viruses of the familyParamyxoviridae, particularly HVJ.

A method of transferring candidate nucleic acids into first host cellsusing a liposome containing at least one protein from viral envelope isalso available. Examples of the protein used in this method include, butare not limited to, F protein, HN protein, NP protein, M protein, or acombination thereof.

The methods for transferring the candidate nucleic acids into first hostcells are also applicable to second host nucleic acids and third hostnucleic acids.

Moreover, after transferring candidate nucleic acids into first hostcells, mutation may be induced in the first host cells to increase thediversity of the nucleic acids transferred in the host cells.Mutagenesis methods for cells are well known, and examples of suchmethods include, but are not limited to, methods using chemicals such asethidium bromide and nitrosoguanidine, and physical methods such as UVirradiation, X-ray irradiation and radioactive ray radiation. In thepresent invention, after increasing the diversity through mutagenesis, acandidate nucleic acid having the intended functional property may beselected from the resultant mutants.

When candidate nucleic acids are expressed in the first host cells, thecandidate nucleic acids may be operably linked with a regulatory elementsuch as promoter. This expression may be transient or stable.

Transient expression of candidate nucleic acids in the first host cellsis sufficient. However, the host cells may allow stable expression ofthe candidate nucleic acids after the transient expression of candidatenucleic acids occurs. Such a host cell is also within the scope of thepresent invention.

Next, from the host cells into which the nucleic acids are transferred,a cell into which a nucleic acid having the intended functional propertyhas been transferred is selected. The intended functional property isselected, for example, from the group consisting of induction ofangiogenesis, tumor suppression, enhancement of osteogenesis, inductionof apoptosis, cytokine secretion, induction of dendrites, suppression ofarteriosclerosis, suppression of diabetes; suppression of autoimmunediseases; suppression of Alzheimer's disease, suppression of Parkinson'sdisease, protection of nerve cells and combinations thereof.

Preferably, this selection is effected based on the phenotype of a hostcell which changes in response to expression of candidate nucleic acids.For example, when a gene encoding a growth factor is isolated fromcandidate nucleic acids, the intended functional property is promotionof growth of a specific cell or any cells.

The method of the present invention may be applicable to any functionalproperties as long as the functional property of interest isrecognizable. Examples of functional properties intended in the presentinvention include, but are not limited to, promotion or suppression ofcell growth; differentiation or de-differentiation of cells; expressionof marker proteins or suppression of the expression of marker proteins;expression of marker mRNA or suppression of expression of marker mRNA;change in membrane potential; depolarization; apoptosis; carcinogenesis;arrest of growth; change in morphology; change in size and the like.

Regarding the method for preparing a purified nucleic acid from a cellthat is selected as containing a nucleic acid having the intendedfunctional property, the second host cell may or may not be used. Whenthe second host cell is used, the method is carried out in the followingsteps without limitation:

(i) extracting a nucleic acid from the selected cell;

(ii) transferring the extracted nucleic acid into a second host cell tothereby obtain a transformed cell;

(iii) purifying the transformed cell; and

(iv) preparing a nucleic acid from the purified, transformed cell.

In the foregoing method, a variety of well-known methods andcommercially available kits may be used to extract nucleic acids fromthe first host cells. Depending on the kind of the second host cell,various well-known methods may be applied so as to transfer the nucleicacid to the second host cell. For example, when a bacterium is used asthe second host cell, gene transfer may be achieved in the followingmanner without any limitation: preparing competent cells by a calciummethod or the like, and transferring nucleic acids into bacterial hostcells by the application of heat shock. Electroporation may also beused. Non-limiting examples of methods for transferring DNA into fungiinclude the use of electroporation [Methods. Enzymol., 194, 182 (1990)],spheroplasts [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], and lithiumacetate. When a plant cell is used as a host cell, known methodsinclude, but are not limited to, the use of Agrobacterium (JapaneseLaid-Open Publication No. 59-140885, Japanese Laid-Open Publication No.60-70080, WO94/00977), electroporation methods (Japanese Laid-OpenPublication No. 60-251887) and methods using a particle gun (gene gun).When an animal cell is used as a host cell, available methods includebut are not limited to, a transferring method using a vital envelope, atransferring method using a liposome, a transferring method using aliposome containing at least one protein from viral envelope, atransferring method using calcium phosphate, and electroporationmethods.

In the case where a second host cell is used, preferably only one kindof candidate nucleic acid per cell is transferred into the second hostcell. Therefore, when a population of candidate nucleic acids isobtained, the population is transferred into the second host cell, and ahost cell having a nucleic acid transferred therein is purified, wherebythe candidate nucleic acid can be purified.

If a plurality of kinds of purified nucleic acids are observed afterpurification of the nucleic acids, a purified nucleic acid having theintended functional property may be selected from the purified nucleicacids. This selection may be achieved by expressing the purified nucleicacid, confirming the function of the nucleic acid, and determiningwhether or not the confirmed function is the intended function.Alternatively, it may be achieved by sequencing the whole or a part ofthe purified nucleic acid structure.

Kits for practicing the methods of the present invention are alsoprovided in the present invention.

(1. Preparation of a Viral Envelope Vector)

Various methods for preparing a viral envelope vector are known in theart. For example, the present inventors developed a hybrid gene transfervector by combining a viral vector and a non-viral vector, andconstructed a fusion forming viral liposome having a fusion formingenvelope derived from hemagglutinating virus of Japan (HVJ: SendaiVirus) (Kaneda, Biogenic Amines, 14:553-572 (1998); Kaneda et al, Mol.Med. Today, 5:298-303 (1999)). In this delivery system, a liposomefilled with DNA is fused with UV inactivated HVJ, to thereby form a HVJliposome which is a fusion forming viral liposome (diameter: 400-500nm). Fusion-mediated delivery is advantageous in that transfection ofDNA is protected from endosomal degradation and lysosomal degradation inthe recipient cell. DNA having a length of up to 100 kb is incorporatedinto an HVJ liposome, and delivered into a mammalian cell. RNA,oligonucleotides and drugs are also introduced into a cell efficientlyin vitro or in vivo. HVJ-liposome was not shown to induce significantcell damage in vivo.

Repeated transfection in vivo has succeeded due to the lowimmunogenicity of HVJ (Hirano et al., Gene Ther., 5:459-464 (1993)).This vector system was modified, and anion type and cation typeHVJ-liposomes have been developed for more efficient gene delivery(Saeki at al., Hum. Gene Ther., 8:1965-1972 (1997)) Using thisHVJ-liposome system, a great number of gene therapy strategies havesucceeded (Dzau et al, Proc. Natl. Acad. Sci. USA, 93:11421-11425(1996);Kaneda et ale, Mol. Med. Today, 5:298-303 (1999)). Several attempts toconstruct HVJ-derived synthetic virosomes have also been made (Wu etal., Neuroscience Lett., 190:73-76 (1995)); Ramani et al., FEBS Lett.,404:164-168 (1997); Ramani et al., Proc. Natl. Acad. Sci. USA,95:11886-11890 (1998)).

When it is necessary to inactivate a virus for preparing a vector, avariety of known methods may be used. Typical inactivation methodsinclude, but are not limited to, UV irradiation, treatment with analkylation agent, treatment with β-propiolacton, treatment withsurfactant, and partial degradation of the envelope by enzymatictreatment.

Modifications to the foregoing methods of preparing a viral envelopevector are also known. Typical examples are shown below. The method ofpreparing a viral envelope vector shown below is only for illustration,and the present invention is not limited to vectors that are prepared inthe method described below.

(1.1. Preparation of a Gene Transfer Vector Encapsulating a Foreign Genein a Component Containing a Protein Derived from Viral Envelope)

Examples of a gene transfer vector containing a protein derived from aviral envelope include, but are not limited to, gene transfer vectorsconsisting of liposomes obtained by reconstitution of the F fusionprotein and HN fusion protein of HVJ (Sendai Virus), but not includingan amount of HVJ genomic RNA that is detectable by RT-PCR.

The F fusion protein and HN fusion protein used in preparation of such agene transfer vector may be protein of naturally-occurring HVJ orrecombinantly expressed protein, Recombinantly produced fusion proteinsare subjected to in vitro processing with proteases, or processing withendogenous proteases in a mammalian host cell.

A gene transfer vector containing a protein derived from a viralenvelope is prepared, for example, by a method comprising the followingsteps:

isolating a fusion protein from HVJ virus that has not been irradiatedwith UV;

reconstituting the fusion protein in the presence of a surfactant and alipid to prepare a reconstituted particle;

preparing a liposome filled with the intended nucleic acid; and

fusing the reconstituted particle and the liposome.

The surfactant used in the above method is not particularly limited to aspecific surfactant, and preferred examples include octylglucoside,Triton-X100, CHAPS or NP-40, or mixtures thereof.

The lipid used in the above method is not particularly limited to aspecific lipid, and may be those (1) having a long chain fatty acid or asimilar hydrocarbon chain in the molecule, and (2) naturally occurringin an organism or derived from an organism. Preferred examples of thelipid include, but are not limited to, phosphatidyl choline,phosphatidyl serine, cholesterol, sphingomyelin, and phosphatidic acid.

A preparation method of liposomes is well known, and for example, thefollowing method may be used:

(A) Prepare a thin film of phospholipids in a first test tube inadvance. Nitrogen gas saturated with water at 55° C. is introduced tothis test tube and allowed to sufficiently hydrate the thin film ofphospholipids. Upon completion of hydration, the thin film turnstransparent.

(B) To the test tube A, a buffer of from a second test tube is addedsmoothly, and after introduction of nitrogen gas, the test tube issealed (for example, with Parafilm) and kept in an incubator (which isan apparatus that enables the experiment to be conducted at a constanttemperature) at 37° C. for about two hours.

(C) Macro-liposomes are prepared by gentle shaking. As a result ofgeneration of liposomes, the liquid becomes slightly cloudy. Using this,the following measurement is conducted.

(D) Placing a droplet of sample on a slide glass, the morphology of theliposomes is observed under a fluorescence microscope (×1000).

A method of preparing a protein that is required for preparing a genetransfer vector encapsulating a foreign gene in a component containing aprotein derived from a viral envelope is well known.

For example, it may be prepared by purification of HVJ envelope proteinfrom naturally occurring HVJ, or purification of recombinantly expressedHVJ envelope protein. Examples of well-known protein purificationmethods include, but are not limited to, ammonium sulfate precipitation,electrofocusing, and purification using a column. When a protein ispurified using a column, various columns may be selected depending onthe properties of the intended protein and the properties of likelycontaminants. Examples of columns used for protein purification include,but are not limited to, an anion exchange column, a cation exchangecolumn, a gel filtration column and an affinity column.

Alternatively, a gene transfer vector containing a protein derived froma viral envelope is prepared by a method comprising the steps of:

recombinantly expressing the F protein and HN protein of HVJ;

processing F protein with a protease;

isolating F protein and HN protein;

reconstituting F protein and HN protein in the presence of a surfactantand lipids to prepare a reconstituted particle;

preparing a liposome filled with nucleic acid; and

fusing the reconstituted particle and the liposome.

A gene transfer vector containing a protein derived from a viralenvelope may also be prepared by a method comprising the steps of:

recombinantly expressing F protein and HN protein, in a host cell inwhich a protease that processes F protein is expressed;

isolating F protein and HN protein;

reconstituting F protein and HN protein in the presence of a surfactantand lipids to prepare a reconstituted particle;

preparing a liposome filled with an intended nucleic acid; and

fusing the reconstituted particle and the liposome.

(1.2. Preparation of a Gene Transfer Vector Encapsulating a Foreign Genein a Viral Envelope)

One exemplary method of preparing a gene transfer vector encapsulating aforeign gene in a viral envelope comprises the following steps:

1) mixing a virus and a foreign gene; and

2) freezing and thawing the mixture, or mixing the mixture with asurfactant.

Alternatively, a gene transfer vector derived from viral envelope can beprepared by a method comprising the steps of:

inactivating a virus;

mixing the inactivated virus with a foreign gene; and

freezing and thawing the mixture.

In a further aspect of the present invention, there is provided a methodof preparing an inactivated virus envelope vector for gene transfer,comprising the steps of:

inactivating a virus; and

mixing the inactivated virus with a foreign gene in the presence of asurfactant.

(1.3. Liposome Vector)

A liposome vector may use liposomes prepared from lipids commonly usedin lipofection. For example, lipids such as lipofect AMINE 2000 may beused.

(Selection of Cells)

In the present invention, a variety of cells may be used as the firsthost cell. The first host cells are preferably mammalian cells and morepreferably cells derived from the species from which the candidatenucleic acid is derived.

In the present invention, a variety of cells may be used as the secondhost cell. Any kind of cells may be used as the second host cell. Cellssuited for the second host cell will incorporate one kind of candidatenucleic acid per cell. Examples of such cells include, but are notlimited to, bacterial and fungal cells.

In the present invention, a variety of cells may be used as the thirdhost cell. The third host cell is preferably the same as the first hostcell, or a cell having a similar gene expression mechanism.

The present invention has now been illustrated with its preferredembodiments. The present invention will further be illustrated based onthe Examples and referring to the drawings attached hereto. It should benoted that the following Examples are provided by way of illustration,and are not intended to limit the present invention. Therefore, thescope of the present invention is not limited to any specificembodiments recited by the examples below, and is only defined by theattached claims.

EXAMPLES Example 1 Preparation and Use of a Gene Transfer VectorEncapsulating a Foreign Gene and a Component Containing a ProteinDerived from Viral Envelope

(Preparation of Virus)

HVJ, Z strain, was purified by differential centrifugation as previouslydescribed (Kaneda, Cell Biology: A Laboratory Handbook, J. E. Cells(Ed.), Academic Press, Orlando, Fla., vol. 3, pp. 50-57 (1994)). Thepurified HVJ was resuspended in a buffered salt solution (BSS: 137 mMNaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH7.5), and the virus titer wasdetermined by measuring absorbance at 540 nm. Optical density at 540 nmcorresponds to 15,000 haemocyte aggregation unit (HAU) and correlateswith fusion activity.

(Extraction of F and HN Fusion Protein from HVJ)

Nonidet P-40 (NP-40) and phenylmethylsulfonyl fluoride (PMSF) dissolvedin ethanol were added to 20 mL of suspension of purified HVJ (1,750,000HAU), at final concentrations of 0.5% and 2 mM, respectively. Themixture was incubated at 4° C. for 30 minutes with mixing. Then, thesuspension was centrifuged at 100,000 g, 4° C. for 75 minutes to removeinsoluble proteins and virus genome (Uchida et al., 1979). Thesupernatant was dialyzed against 5 mM phosphate buffer (pH 6.0) forthree days, and the remaining NP-40 and PMSF were removed off byexchanging the buffer every day. The dialyzed solution was centrifugedat 100,000 g, 4° C. for 75 minutes, thereby removing insolublesubstances. The supernatant was applied to an ion exchange column ofCM-Sepharose CL6B (Pharmacia Fine Chemicals, Uppsala, Sweden) that wasequilibrated with 10 mM phosphate buffer (pH 5.2) containing 0.3M-sucrose and 1 mM KCl, according to the previously described method(Yoshima et al., J. Biol. Chem., 256:5355-5361 (1981)). Flow-throughfractions and the 0.2 M NaCl eluate were collected. These fractions weresubjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and analyzed for protein components. The gel was stained withCoomassie brilliant blue, and the ratio of each protein was evaluatedusing computerized densitometry (NIH Image; Apple Computers, Cupertino,Calif., USA).

(Recombinant Expression)

A fusion protein of HVJ may be prepared by incorporating a gene codingthe fusion protein into an expression vector and expressing the gene inan appropriate host cell. The amino acid sequences of F protein and HNprotein are known.

Expression vectors that may be used in various host cells arecommercially available.

An expression vector encoding a fusion protein that may be transferredinto a cell and subsequent production of a fusion protein of the presentinvention is known in the art, and can be carried out by any of thevarious described methods (for example, Sambrook et al., MolecularCloning; A Laboratory Manual, 2nd Ed, Vols. 1 to 3, Cold Spring HarborLaboratory Press, New York (1989) and Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1994), eachincorporated herein by reference)). Examples of methods for transferringa recombinant expression vector into a prokaryotic or a eukaryotic cellinclude electroporation methods, transformation methods, or transfectionmethods.

When the recombinant F protein was expressed in Escherichia coli, it wasexpressed in an inactive F0 form. In order to convert the inactive F0form of protein expressed in Escherichia coli into an active F1 form,trypsin treatment at 37° C. for 30 minutes using 0.0004-0.001% trypsinwas required.

The polypeptide corresponding to activated F1 protein treated withtrypsin may be expressed in Escherichia coli using an expression vectorcontaining a gene encoding the amino acid sequence of a truncated,activated F1. The truncated F1 protein necessarily includes at least 26amino acids from phenylalanine, at position 117, to alanine, at position142. In the case where the truncated protein forms an inclusion body,those skilled in the art can readily obtain an active form of theprotein by refolding the inclusion body (see Robert F. Kelley andMarjorie E. Winkler, Genetic Engineering, (1990) vol. 12, pp. 1-19 forreference).

When F protein is expressed by using cells in which HVJ can replicate(for example, rodent tracheal epithelium; chicken embryos; f monkeykidney primary culture cells; human fetal lung primary culture cells,kidney and amnion) as a host cell, the expressed full length F proteinis cleaved by an endogenous protease of the host cell, and as a result,is activated. In this manner, an active form of F protein can beexpressed and isolated. Alternatively, host cells in which Tryptaseclara (Kido et al., Molecular Cells 9, 235-244 (1999)) is expressed asan endogenous enzyme (for example, rat tracheal epithelium) or hostcells in which Tryptase clara is recombinantly expressed may also beused.

Methods of selecting and constructing these expression vectors, methodsof transferring into host cells, methods of expressing in host cells,and methods of collecting expressed proteins are well known by thoseskilled in the art.

(Purification of Fusion Protein from HVJ)

In order to purify a fusion protein, a lysate of HVJ treated with NP-40was clarified by ultracentrifugation. Proteins in the supernatant wereanalyzed by SDS-PAGE prior to further purification. This supernatantcontained many proteins derived from HVJ. Then, the supernatant wasapplied to an ion exchange column. Proteins of 52 kDa and 72 kDa weredominantly eluted in the flow-through fraction. These two proteins wereidentified as F1 and HN, respectively, based on their mobility in anSDS-PAGE gel (Okada, Methods in Enzymology, N. Duzgnes (Ed.), AcademicPress; San Diego, vol. 221, pp. 18-41 (1993)). A minor band under the 52kDa protein was considered to be a degradation product of the fusionproteins (F1 and HN). This is because these proteins were notreproducibly observed in different experiments. Proteins were furthereluted with 0.2 M NaCl. However, fusion proteins were not obtainedefficiently. Furthermore, a protein of 60 kDa that is speculated to beNP protein of HVJ additionally appeared. Eventually, only theflow-through fraction was used as a source of fusion proteins forsubsequent experiments. Densitometry indicated that the F1 to HNconcentration ratio in the flow-through fraction was 2.3:1. This isconsistent with the ratio of these proteins in the viral envelope. Aprevious paper (Nakanishi et al., Exp. Cell Res., 142:95-101 (1982))reported that this ratio is required for efficient fusion of HVJ.

(Preparation of a Gene Transfer Vector)

A lipid mixture consisting of 3.56 mg of phosphatidyl choline and 0.44mg of cholesterol was dissolved in chloroform, and the resultant lipidsolution was evaporated in a rotary evaporator (Uchida et al., J. Cell.Biol. 80:10-20 (1979)). The dry lipid mixture was completely dissolvedin 2.0 mL of protein solution (1.6 mg) from the above flow-throughfraction containing 0.85% NP-40 using a Vortex mixer.

This solution was then dialyzed against 10 mM phosphate buffer (pH 7.2)containing 0.3 M sucrose and 1 mM KCl to thereby remove NP40. Thedialysis was continued for 6 days, and the buffer was replaced everyday. The dialyzed solution was applied to agarose beads (Bio-GelA-50m)(Bio-Rad Laboratories, Hercules, Calif., USA) and equilibrated with 10mM phosphate buffer (pH5.2) containing 0.3 M sucrose and 1 mM KCl.Fractions having an optical density of more than 1.5 at 540 nm werecollected as reconstituted fusion particles. The gene transfer vectorwas prepared by fusing the reconstituted fusion particles with aliposome filled with nucleic acid prepared from 1.0 mg of lipids asdescribed below.

(Expression of Luciferase Gene in HEK293 Strain-Derived TransfectedCells)

In order to confirm the gene transferring activity of the gene transfervector prepared in the aforementioned manner, HEK293 cells and aluciferase gene were used in the following manner.

pCMV-luciferase (7.4 kb) was constructed by cloning a luciferase genefrom pGEM-luc (Promega Corp., Madison, Wis., USA) into pcDNA3 (5.4 kb)(Invitrogen, San Diego, Calif., USA) at Hind III and Bam HI sites. Agene transfer vector containing about 40 μg of pCMV-luciferase wasconstructed in the manner described above, and 1/10 amount (100 μL) ofthe gene transfer vector (about 1.5×10¹¹ particles/mL, DNA concentrationabout 40 μg/mL) was incubated with 2×10⁵ cells derived from human 293cell line (human embryonic kidney: HEK). Using HVJ liposomes, the sameamount of luciferase DNA was transferred into 2×10⁵ HEK293 cells. Twentyfour hours after transduction, the cells were collected, and assayed forluciferase activity in the described manner (Saeki et al., Hum. GeneTher., B: 1965-1972 (1997)).

Example 2 Preparation of HVJ Envelope Vector by Freezing and Thawing andUses Thereof

(Preparation and Use of a Gene Transfer Vector)

(1: Preparation of HVJ Envelope Vector by Freezing and Thawing)

A recombinant HVJ virus containing a luciferase gene as a foreign gene,was subjected to various cycles of freezing and thawing before beingtransferred into a cultured cell.

Five hundred μL of TE, 750 μg of luciferase expression vector pcOriPLuc(Saeki and Kaneda et al., Human Gene Therapy, 11, 471-479 (2000)) andvarious concentrations of HVJ virus were mixed. HVJ virus was preparedin concentrations of 10, 25, 50 and 100 HAU/μL. The resultant solutionwas divided into 12 aliquots, and each aliquot was subjected to up to 30cycles of freezing and thawing, each cycle consisting of freezing bystoring at 4° C., frozen with dry ice and thereafter thawed; this wasrepeated up to 30 times. The resultant solution, having been subjectedto a predetermined number of freezing and thawing cycles, was added tothe medium of BHK-21 cells (4×10⁴ cells/dish, 0.5 mL DMEM, 10% FCS per24-well dish), allowed to react at 37° C. under 5% CO₂ for 20 minutes,the cells were washed with PBS, and then 0.5 mL of culture medium wasfreshly added and the cells cultured for 24 hours.

After removing the medium, 500 μL of 1×Cell Culture Lysis Reagent(Promega) was added to the cells to lyse the cells, and the resultingcell suspension was centrifuged in a micro tube. Twenty μL of theobtained supernatant was measured for luciferase activity using thePromega Luciferase Assay System and Lumat LB9501 Luminophotometer. Themeasurement was conducted three times for each solution, and an averagevalue was determined.

As a result, it was observed that luciferase activity increased with thenumber of cycles of freezing and thawing of the recombinant HVJ virus.Upon 20 cycles of freezing and thawing, ten fold or more luciferaseexpression was observed as compared to 3 cycles of freezing and thawing.This result revealed that the number of cycles of freezing and thawingof recombinant HVJ virus is preferably 5 or more, more preferably about15 to 20 under the conditions of the present Example.

(2: Gene Transferring Efficiency of HVJ Envelope Vector Prepared byFreezing and Thawing)

After 30 cycles of freezing and thawing of the recombinant HVJ virus,similar to that shown in Example 1 above, the transfer efficiency of agene into cells was examined under the condition wherein the number ofviruses added to the host cells was constant.

For example, in the case where the X axis is 500 HAU, 50 μL of asolution having a virus concentration of 10-50 HAU/μL, and 5 μL of asolution having a virus concentration of 100 HAU/μL were used. Theefficiency of gene expression for a solution having a virusconcentration of 100 HAU/μL was lower by about 50% compared with thatfor a solution having a virus concentration of 10-50 HAU/μL. This resultrevealed that under the conditions of this example, the recombinantvirus concentration was preferably in the range of 10 to 50 HAU/μL.

After 29 cycles of freezing and thawing of the recombinant HVJ virus,the 30th freezing was conducted, the recombinant virus stored frozen forone week and then thawed before being added to cells. The recombinantHVJ virus stored frozen for one week and the recombinant HVJ virussubjected to a continuous 30 cycles of freezing and thawing showedsimlar levels of luciferase gene expression.

Example 3 Preparation of an Inactivated HVJ Envelope Vector Utilizing aDetergent

(1: Growth of HVJ)

In general, HVJ cultured by inoculating a fertilized chicken egg withseed virus may be used. However, HVJ grown in cultured cells (e.g.,simian or human) or a persistent infection system (i.e., a culturemedium supplemented with a hydrolase such as trypsin is added tocultured tissue), or HVJ grown by infecting cultured cells with clonedvirus genome to cause persistent infection are applicable.

In the present example, the growth of HVJ was performed as follows.

HVJ seed virus was cultured in a SPF (Specific Pathogen Free) fertilizedegg. The isolated and purified HVJ (Z species) was dispensed into acryo-vial, DMSO added to 10%, and stored in liquid nitrogen.

Chicken eggs were obtained immediately after fertilization, and placedin an incubator (SHOWA-FURANKI P-03 type; capable of accommodating about300 chicken eggs), and incubated for 10 to 14 days at 36.5° C. and 40%or more humidity. In a darkroom, the viability of the embryo as well asthe air cell and the chorioallantoic membrane was confirmed using an eggtester (specifically, an egg-tester in which light from a light bulb isprojected through a window having a diameter of about 1.5 cm). Avirus-injection site was marked in pencil about 5 mm above thechorioallantoic membrane (the position was selected so as to avoid anythick blood vessels). The seed virus (which was removed from liquidnitrogen) was diluted 500-fold with a polypeptone solution (1%polypeptone, 0.2% NaCl, adjusted to pH 7.2 with 1 M NaOH, thenautoclave-sterilized and stored at 4° C.), and left at 4° C. The egg wasdisinfected with Isodine™ and alcohol. A small hole was made in thevirus-injected site with a pick. Using a 1 ml syringe and a 26 gaugeneedle, 0.1 ml of the diluted seed virus was injected into thechorioallantoic cavity. Molten paraffin (melting point: 50 to 52° C.)was placed onto the hole using a Pasteur pipette in order to seal thehole. The egg was placed in an incubator and incubated for three days at36.5° C. and 40 or more humidity. The inoculated egg was then leftovernight at 4° C. The following day, the air cell portion of the eggwas broken with forceps, and a 10 ml syringe with an 18 gauge needle wasplaced in the chorioallantois so as to aspirate the chorioallantoicfluid, which was collected in a sterilized bottle and stored at 4° C.

(2: Purification of HVJ)

HVJ may be purified by purification methods utilizing centrifugation,purification methods utilizing a column, or any other purificationmethods known in the art.

(2.1: Centrifugation-Based Purification Method)

Briefly, a suspension of cultured viruses was collected, and the mediumcentrifuged at low speed to remove tissue or cell debris in the culturemedium and the chorioallantoic fluid. The supernatant thereof waspurified by high-speed centrifugation (27,500×g, 30 minutes) andultracentrifugation (62,800×g, 90 minutes) on a sucrose density gradient(30 to 60% w/v). Care should be taken to treat the virus as gently aspossible during purification, and to store the virus at 4° C.

Specifically, in the present example, HVJ was purified by the followingmethod.

About 100 ml of HVJ-containing chorioallantoic fluid (thechorioallantoic fluid from chicken eggs containing HVJ, which wascollected and stored at 4° C.) was placed in two 50 ml centrifuge tubeswith a wide-mouth Komagome type pipette (see Saeki, Y., and Kaneda, Y:Protein modified liposomes (HVJ-liposomes) for the delivery of genes,oligonucleotides and proteins. Cell Biology; A laboratory handbook (2ndedition) ed. by J. E. Celis (Academic Press Inc., San Diego) vol. 4, 127to 135,1998), centrifuged in a low-speed centrifuge at 3000 rpm and at4° C. for 10 minutes (without braking) to remove the tissue debris fromthe egg.

After centrifugation, the supernatant was dispensed into four 35 mlcentrifuge tubes (designed for high-speed centrifugation), andcentrifuged for 30 minutes in a fixed-angle rotor at 27,000 g, (withacceleration and braking). The supernatant was removed, BSS (10 mMTris-HCl (pH 7.5), 137 mM NaCl, 5.4 mM KCl; autoclaved and stored at 4°C.) (BSS is interchangeable with PBS) was added to the pellet in anamount of about 5 ml per tube, and allowed to stand at 4° C. overnight.The following morning, the pellets were resuspended by gentle pipettingwith a wide-mouth Komagome type pipette and collected in one tube, andthen similarly centrifuged for 30 minutes in a fixed-angle rotor at27,000 g. The supernatant was removed, and about 10 ml of BSS was addedto the pellet and allowed to stand at 4° C. overnight. The followingmorning the pellets were resuspended by gentle pipetting with awide-mouth Komagome type pipette and then centrifuged for 10 minutes ina low-speed centrifuge at 3000 rpm at 4° C. (without braking), therebyremoving tissue debris and agglutinated virus which had not beencompletely removed. The supernatant was placed in a fresh sterilizedtube, and stored at 4° C. as the purified virus stock.

To 0.1 ml of this virus solution, 0.9 ml of BSS was added, and theabsorption at 540 nm was measured with a spectrophotometer. The virustiter was converted into an erythrocyte agglutination activity (HAU). Anabsorption value of 1 at 540 nm approximately corresponded to 15,000HAU. It is considered that HAU is substantially proportional to fusionactivity. Alternatively, erythrocyte agglutination activity may bemeasured by using a solution containing (0.5%) chicken erythrocytes (seeDOUBUTSU SAIBO RIYO JITSUYOKA MANUAL (or “Practice Manual for UsingAnimal Cells”), REALIZE INC. (ed. by Uchida, Oishi, Furusawa) pp. 259 to268, 1984).

Furthermore, purification of HVJ using a sucrose density gradient may beperformed as necessary. Specifically, a virus suspension is placed in acentrifuge tube in which 60% and 30% sucrose solutions(autoclave-sterilized) are layered, and the density gradient centrifugedfor 120 minutes at 62,800×g. After centrifugation, the virus is visibleas a band at the interface of the 60% sucrose solution layer, and isrecovered. The recovered virus suspension is dialyzed overnight at 4° C.against an external solution of BSS or PBS, thereby removing thesucrose. In the case where the virus suspension is not to be immediatelyused, glycerol (autoclave-sterilized) and a 0.5 M EDTA solution(autoclave-sterilized) are added to the virus suspension so as to attainfinal concentrations of 10% and 2 to 10 mM, respectively, the suspensionis then gently frozen at −80° C., and finally stored in liquid nitrogen(the frozen storage can be performed with 10 mM DMSO, instead ofglycerol and a 0.5 M EDTA solution).

(2.2: Purification Method Utilizing Columns and Ultrafiltration)

Instead of purification by centrifugation, purification of HVJ utilizingcolumns is also applicable to the present invention.

Briefly, concentration (about 10 times) via ultrafiltration utilizing afilter having a molecular weight cut-off (MWCO) of 50,000 and elutionvia ion exchange chromatography (0.3 M to 1 M NaCl) were performed toachieve purification.

Specifically, in the present example, the following method was used topurify HVJ.

After the chorioallantoic fluid was collected, the chorioallantoic fluidwas filtrated through a membrane filter (80 μm to 10 μm). To thechorioallantoic fluid, 0.006 to 0.008% BPL (final concentration) wasadded (4° C., 1 hour), so as to inactivate the HVJ. The chorioallantoicfluid was incubated for 2 hours at 37° C., thereby inactivating the BPL.

About 10 times concentration was achieved using a 6 tangential flowultrafiltration method using a 500KMWCO membrane (A/G Technology,Needham, Mass.). As a buffer, 50 mM NaCl, 1 mM MgCl₂, 2% mannitol, and20 mM Tris (pH 7.5) were used. An HAU assay indicated an HVJ yield ofapproximately 100%. Thus, excellent results were obtained.

HVJ was purified by column chromatography (buffer: 20 mM Tris HCl (pH7.5), 0.2 to 1 M NaCl) using a Q Sepharose FF column (Amersham PharmaciaBiotech KK, Tokyo). The yield was 40 to 50%, and the purity was 99% ormore.

An HVJ fraction was concentrated by tangential flow ultrafiltrationusing a 500KMWCO membrane (A/G Technology).

(3: Inactivation of HVJ)

In the case where it was necessary to inactivate HVJ, this was performedby UV light irradiation or treatment with an alkylating agent, asdescribed below.

(3.1: UV Light Irradiation Method)

One milliliter of HVJ suspension was placed in a dish having a diameterof 30 mm, and subjected to an irradiation at 99 or 198 mJ/cm². Althoughgamma-ray irradiation is also applicable (5 to 20 Gv), it does notprovide complete inactivation.

(3.2: Treatment with an Alkylating Agent)

Immediately before use, 0.01% β-propiolactone was prepared in 10 mMKH₂PO. The solution was kept at a low temperature during preparation,and the operation was quickly performed.

β-propiolactone was added to a final concentration of 0.01% to the HVJsuspension obtained immediately after purification, and the mixture wasthen incubated on ice for 60 minutes. Thereafter, the mixture wasincubated at 37° C. for 2 hours. The mixture was dispensed intoEppendorf tubes in 10,000 HAU aliquots, and centrifuged for 15 minutesat 15,000 rpm. The precipitate was stored at −20° C. Instead of usingthe aforementioned inactivation method, without storing the precipitateat −20° C., DNA may be incorporated into a vector by detergent treatmentalone when constructing a vector.

(4: Construction of an HVJ Envelope Vector)

To the HVJ which had been stored, 92 μl of a solution containing 200 to800 μg of exogenous DNA was added, and well mixed by pipetting. Thissolution can be stored at −20° C. for at least 3 months. By addingprotamine sulfate to the DNA before mixing with HVJ, the expressionefficiency was enhanced twofold or more.

This mixture was placed on ice for 1 minute, and 8 μl of (10%)octylglucoside was added. The tube was shaken on ice for 15 seconds, andallowed to stand on ice for 45 seconds. The treatment time with thedetergent is preferably 1 to 5 minutes. Instead of octylglucoside,detergents such as Triton-X100 (t-octylphenoxypolyethoxyethanol), CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), or NP-40(nonylphenoxy polyethoxy ethanol) may also be used. The finalconcentrations of Triton-X100, NP-40, and CHAPS are preferably0.24-0.80% (v/v), 0.04-0.12% (v/v) and 1.2-2.0% (v/v), respectively.

One milliliter of cold BSS was added, and the solution was immediatelycentrifuged for 15 minutes at 15,000 rpm. To the resultant precipitate,300 μl of PBS or saline, etc., was added, and the precipitate suspendedby vortexing or pipetting. The suspension may be directly used for genetransfer or may be used for gene transfer after storage at −20° C. Afterbeing stored for at least 2 months, this HVJ envelope vector maintainedthe same level of gene transfer efficiency.

(Gene Transfer Method)

An amount of vector equivalent to 1,000 HAU (30 μl) was placed into anEppendorf tube, and 5 μl of protamine sulfate (1 mg/ml) was added. Themedium was removed from BHK-21 cells (which were sown in 6 well dishesat a density of 200,000 cells per well the previous day) and 0.5 ml ofmedium (10% FCS-DMEM) was added to per well. To each well, a mixture ofthe aforementioned vector (equivalent to 1,000 HAU) and protaminesulfate was added, and the plate was shaken back and forth and fromright to left, whereby the vector and cells were well mixed. The mixturewas left in a 5% CO₂ incubator for 10 minutes at 37° C.

The medium was replaced, and the cells were left overnight (16 hrs to 24hrs) at 37° C. in a 5% CO₂ incubator, after which the gene expressionwas examined. To measure luciferase activity (pcLuci: a luciferase genehaving a CMV promoter), the cells were lysed with 0.5 ml of Cell LysisBuffer (Promega), and the activity in 20 μl of the solution was measuredusing a luciferase assay kit (Promega). To measure green fluorescentprotein activity (pCMV-GFPE; Promega), the cells were observed under afluorescent microscope in their intact form, and 5 to 8 fields wereobserved at a magnification of 400, and the ratio of cells whichgenerated fluorescence was calculated.

Example 4 Preparation and Use of a Liposome Vector)

(Preparation of Liposome Vector)

A liposome vector of the present invention is prepared as follows:Twenty to twenty four μg of cDNA library-derived cDNA and 24-72 μL oflipofect AMINE 2000 reagent (Invitrogen life technologies (Carlsbad,Calif. 92008) are respectively diluted in 1.2 mL of serum free medium,and rapidly mixed. The mixture is incubated at room temperature for 20minutes, to form a complex of liposomes and nucleic acid.

(Transfection by Liposome Vector)

Host cells are added to each well of 96-well plate together with anappropriate medium, and cultured. When transfection is conducted in thepresence of serum, 12.5 μL of the transfection complex is directly addedto each well of the 96-well plate and mixed. When transfection isconducted in absence of serum, the medium containing serum is removedand replaced by a serum free medium before adding the transfectioncomplex. After incubation in a CO₂ incubator for 4 to 12 hours, themedium is replaced. After a predetermined period of culture, an assay isconducted.

Example 5 Isolation and Analysis of a Gene Encoding Vascular EndothelialGrowth Factor from a Human Heart cDNA Library

Using the present invention, it is possible to isolate a gene ofinterest having an intended functional property. One embodiment of theisolation method is schematically shown in FIG. 1.

Actually, using the gene transfer vector prepared in Example 3 of thisspecification, a gene encoding vascular endothelial growth factor wasisolated. For isolating the gene exemplarily shown in this example, notonly the gene transfer vector prepared in Example 3 of thisspecification, but also any “viral envelope vector” and “liposomevector” may be used.

Human heart cDNA library (GIBCO BRL; plasmid prepared by ligating humanheart-derived cDNA to plasmid pSPORT having a CMV promoter) wastransferred into E. coli DH12S, and the plasmid was prepared from the E.coli. Two hundred μg of plasmid was encapsulated in 10000 HAU of HVJ-Egene transfer vector (gene transfer vector prepared in Example 3 of theinvention, 3×10⁹ particles). About 5000 human aortal endothelial cells(HAEC) (Sanko Junyaku) were added to each well of a 96-well micro titerplate together with growth medium and cultured overnight. The culturedcells were used as host cells. To each well containing host cells, 1/100amount of the above HVJ-E was added. The wells were kept at 37° C. for30 minutes, and then the medium was replaced.

The medium used was a low nutrient condition medium having a serumconcentration of 1%. Under these conditions, culture was conducted forone week. Under these conditions, growth of HAEC was not observed.

After two weeks, a cell growth assay was conducted. Using Cell Titer•96(Promega) as a regent, cell growth was evaluated based on the colorchange that is indicative of the redox state of mitochondria. The resultis shown in FIG. 2.

In FIG. 2, the wells having the deepest color are wells where cellgrowth occurred most actively. The entire microtiter plate was read witha plate reader, and cell growth was graphically shown with a computer asshown in FIG. 3. DNA was extracted from cells in the two wellsexhibiting the greatest growth according to the graph, using a DNeasyTissue Kit available from Qiagen. Since the prepared nucleic acidincludes plasmid DNA, it was transferred into competent E. coli (DH5α;TAKARA) by heat shock.

This E. coli was inoculated on an ampicillin-containing solid media, andallowed to form colonies. From DNA prepared from a single well, about20-200 colonies were obtained. Plasmid DNA (pDNA) was extracted fromeach colony, and the presence of a gene fragment in the plasmid wasconfirmed by restriction enzyme analysis. About 60-70% of plasmids inthe prepared plasmids were plasmids had an insert (the white arrow inFIG. 4 shows an insert fragment).

Next, plasmid DNA was purified using Endo Free Plasmid Maxi Kitavailable from Qiagen, and the purified plasmid was encapsulated inHVJ-E and transferred into HAEC cells again, and a cell growth testsimilar to that described above was conducted. In this cell growth test,the plasmid exhibiting significantly high cell growth is a candidateplasmid that is expected to include a nucleic acid encoding vascularendothelial growth factor. In the present example, two clones (p3743,p77421) exhibited high HAEC growth activity with high reproducibility.Results from one of these clones is shown in FIG. 5.

The gene products isolated in the present experiment had higher growthactivity than VEGF or HGF with respect to human aortal endothelial cellsHAEC. However, they exhibited similar activity with VEGF or HGF withrespect to human vascular smooth muscle cells.

(Discussion of Angiogenetic Activity)

The foregoing two genes exhibiting high HAEC growth activity (p3743,p77421) were evaluated for angiogenetic ability using an AngiogenesisKit, KZ-1000 (KURABO INDUSTRIES LTD.) in accordance with the followingprocedure. As controls, blank, vascular endothelial growth factor-Aprotein (VEGF-A), pVEGF plasmid (a plasmid containing a gene encodingvascular endothelial growth factor) and pSPORT1 (a plasmid notcontaining a gene encoding vascular endothelial growth factor) wereused. Each obtained image was quantified using an angiogenesisquantification software (KSW-5000U, KURABO INDUSTRIES LTD.) inaccordance with the following procedure.

On a 24-well plate, an angiogenesis KIT KZ-1000 (KURABO INDUSTRIES LTD.)co-culture human umbilical vein endothelial cell and human adultskin-derived fibroblast was used. To a medium specified for angiogenesis(KZ-2400, KURABO INDUSTRIES LTD.) 10 ng/mL of VEGF-E (NZ-7) was added,and anti human VEGF-A neutralizing antibody was added in concentrationof 0, 250, 500, 1000 ng/mL and the medium used to culture the abovecells.

Anti-VEGF, Human, Mouse-Mono (26503.111) (R&D, Catalog No. MAB293) wasused as the anti human VEGF-A neutralizing antibody. Culture wasconducted at 37° C. in a 5% CO₂ incubator. After 4, 7 and 9 days ofculture, the medium was replaced with fresh media supplemented with thesame additives. After 11 days of culture, the medium was removed, andstaining was conducted using a lumen staining kit (for staining CD31antibody: KURABO INDUSTRIES LTD. KZ-1225) in accordance with thefollowing procedure.

CD31 (PECAM-1)-staining primary antibody (mouse anti-human CD31antibody) was 4,000-fold diluted in blocking solution (Dulbeccophosphate buffer (PBS(−) containing 1% BSA). To each well, 0.5 mL ofthis primary antibody solution was added, and incubated for 60 minutesat 37° C. After incubation, each well was washed a total of three timeswith 1 mL of blocking solution.

Then, 0.5 mL of a secondary antibody solution (goat anti-mouse IgGalkaline phosphatase complex) that was 500-fold diluted with a blockingsolution was added to each well, incubated for 60 minutes at 37° C., andthen washed three times with 1 mL of distilled water. During this, twotablets of BCIP/NBT were dissolved in 20 mL of distilled water, andfiltered through a filter having a pore size of 0.22 μm, to prepare asubstrate solution. Then 0.5 mL of the prepared BCIP/NBT solution wasadded to each well, and incubated at 37° C. until the lumen turned deepviolet (usually 5 to 10 minutes). After completion of incubation, eachwell was washed three times with 1 mL of distilled water, and thewashing solution removed by aspiration. Then each well was left to standin order to air dry. After drying, each well was observed under amicroscope.

Each well was observed under ×40 magnification, and photographed.

A picture in which a scale of 1 mm magnified 40-folds was taken (FIG.6), and based on this scale, the area of the lumen (left in FIG. 7), thelength of the lumen (right in FIG. 7), the joints of the lumen (left inFIG. 8) and the path of the lumen (right in FIG. 8) formed in eachvisual field were measured. The number of branch points of the lumen isdenoted by “joint” and the number of lumens coming from the branch pointis denoted by “path”.

The observation result shown in FIG. 6 and the date of the lumen formedshown in FIGS. 7 and 8 demonstrated that clone p77421 has a similardegree of angiogenetic activity to VEGF, and the clone p3743 has betterangiogenetic activity than VEGF.

(c-fos Luciferase Assay)

The influence that the product of the genes isolated in the foregoingexperiment exerts on the activity of promoter of c-fos gene was examinedby reporter assay using a reporter plasmid incorporating a c-fos genepromoter upstream of a luciferase gene.

Specifically, the assay was conducted in the following manner.

Endothelial cells were seeded in a 6-well plate, and transfected with ac-fos-luciferase reporter gene (p2FTL) using lipofect AMINE PLUS(GIBCO-BRL). This fos-luciferase reporter gene consists of 2 copies ofthe c-fos 5′-regulatory enhancer element (−357 to −276), the thymidinekinase gene promoter of herpes simplex virus (−200 to +70), and aluciferase gene. It was co-transfected with p3743 plasmid as necessary.Twenty four hours after transfection, transfected cells were incubatedin a serum free medium for 24 hours. As necessary, cells in rest statewere treated with 100 ng/ml of HGF (hepatocyte growth factor) or withGFP (green fluorescent protein) for four hours. After washing with PBSand adding with 500 μL of cell lysis buffer, the cells were kept at roomtemperature for 15 minutes, and thereby lysed. 10 μL of cell extractobtained by lysing cells was mixed with 100 μL of luciferase assayreagent, and light emission was measured for 30 seconds using aluminometor in units of RLU. The significant increase in luciferaseactivity demonstrated that p3743 increases c-fos gene promoter activity(FIG. 9).

The above result demonstrates that a gene having an intended functionalproperty may be readily and conveniently isolated by using the presentinvention.

Example 6 Isolation Method of Mutant Nucleic Acid Having an IntendedFunctional Property

With the present invention, it is possible to isolate a mutant genehaving an intended functional property.

Using a gene transfer vector prepared in Example 3 of thisspecification, a gene encoding vascular endothelial growth factor isisolated. For isolating the gene exemplarily shown in this example, notonly the gene transfer vector prepared in Example 3 of thisspecification, but also any “viral envelope vector” and “liposomevector” may be used.

As a starting material, a nucleic acid comprising a specified gene isselected. A plasmid in which the selected nucleic acid is operablylinked to a sequence that functions as a promoter in a first host cellis constructed. The plasmid is transferred into a first host cellaccording to Example 3.

The first host cell into which the nucleic acid is transferred issubjected to mutagenesis, and about 5000 cells are added on each well of96-well micro titer plate together with a medium, followed by overnightcultivation. After cultivation, mutated host cells in each well arescreened for an intended function. Cells having an intended function areisolated, and nucleic acid is extracted from the cells in the wellexhibiting the most desired property using a DNeasy Tissue Kit availablefrom Qiagen. Since the prepared nucleic acid includes plasmid DNA, it istransferred into competent E. coli (DH5α; TAKARA) by heat shocktransformation.

This E. coli is inoculated onto ampicillin-containing solid media, andallowed to form colonies. From DNA prepared from a single well, about20-200 colonies can be obtained. Plasmid DNA is extracted from eachcolony, and the presence of a gene fragment in the plasmid is confirmedby restriction enzyme digestion.

Then, using an Endo Free Plasmid Maxi Kit available from Qiagen, plasmidDNA is purified, and the purified plasmid is encapsulated in HVJ-E andtransferred into HAEC cells again, and a similar functional property isexamined. In this cell growth test, the plasmid exhibiting a preferredfunctional property is recognized as a plasmid containing a mutantnucleic acid having an intended functional property.

Although the present invention has been described in its preferredembodiments, it should be noted that the present invention is notlimited to these embodiments. Thus, it can be understood that the scopeof the present invention is defined only by the claims attached hereto.It can be understood that those skilled in the art can practice anequivalent scope based on the description of the present invention andthe common general knowledge in the art in view of the description ofthe specific preferred embodiments. It is appreciated that patents,patent applications and documents cited in this specification areincorporated herein by reference as if the content thereof isspecifically described in the present specification.

INDUSTRIAL APPLICABILITY

A novel method and a kit for isolating a nucleic acid having an intendedfunctional property are provided. As a result, it is possible to isolatea nucleic acid having an intended functional property more rapidly andconveniently than the conventional method. The present invention isparticularly effective for expression screening using mammalian cells asa host cell.

In the present invention, by changing the combination of an objectivecell and an assay method, it is possible to isolate genes having avariety of different functions. Specific examples of such genes includetumor-suppressor genes; osteogenesis enhancer genes; apoptosis triggergenes; cytokine secretion genes; nerve cell dendrite inducer genes;arteriosclerosis suppressor genes; diabetes suppressor genes; autoimmunediseases suppressor genes; Alzheimer's disease suppressor genes;Parkinson's disease suppressor genes and nerve cell protecting genes andthe like.

1. A method of isolating a nucleic acid having an intended functionalproperty, comprising the steps of: (A) transferring a nucleic acid intoa plurality of first host cells and allowing the nucleic acid totransiently express therein; (B) selecting, from the plurality of firsthost cells into which the nucleic acid is transferred, a cell into whicha nucleic acid having an intended functional property has beentransferred; (C) preparing a purified nucleic acid from the selectedcell; and (D) selecting a purified nucleic acid having an intendedfunctional property.
 2. The method according to claim 1, wherein atleast two kinds of nucleic acids are transferred into the plurality offirst host cells.
 3. The method according to claim 1, wherein the stepof transferring a nucleic acid into the plurality of first host cells iscarried out according to a procedure selected from the group consistingof: a transferring method using a viral envelope, a transferring methodusing a liposome, a transferring method using a liposome containing atleast one protein from a viral envelope, a transferring method usingcalcium phosphate and an electroporation method.
 4. The method accordingto claim 1, wherein the nucleic acid includes a foreign gene and apromoter.
 5. The method according to claim 1, wherein the host cells aremammalian cells.
 6. The method according to claim 1, wherein the hostcells are human cells.
 7. The method according to claim 1, wherein theviral envelope is derived from wild-type or recombinant viruses.
 8. Themethod according to claim 1, wherein the viral envelope is derived froma virus belonging to a family selected from the group consisting ofRetroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae,Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxyiridae,Herpesviridae, Baculoviridae and Hepadnaviridae.
 9. The method accordingto claim 8, wherein the virus is derived from viruses belonging to thefamily Paramyxoviridae.
 10. The method according to claim 9, wherein thevirus is HVJ.
 11. The method according to claim 1, wherein the vector isa viral envelope vector.
 12. The method according to claim 1, whereinthe vector is a vector containing a protein prepared from a viralenvelope and a liposome.
 13. The method according to claim 12, whereinthe protein prepared from a viral envelope is a protein selected fromthe group consisting of F protein, HN proteins NP protein and acombination thereof.
 14. The method according to claim 1, wherein thestep (C) of preparing a purified nucleic acid from the selected cell iscarried out in the following steps of: (i) extracting a nucleic acidfrom the selected cell; (ii) transferring the extracted nucleic acidinto a second host cell to thereby obtain a transformed cell; (iii)purifying the transformed cell; and (iv) preparing a nucleic acid fromthe purified transformed cell.
 15. The method according to claim 14,wherein the second host cell is a bacterium or fungus.
 16. The methodaccording to claim 15, wherein the nucleic acid contains a sequence thatis necessary for autonomous replication in the bacterium or fungus. 17.The method according to claim 15, wherein the bacterium belongs to agenus selected from the group consisting of Escherichia, Bacillus,Streptococcus, Staphylococcus, Haemophilus, Neisseria, Actinobacillusand Acinetobacter.
 18. The method according to claim 17, wherein thebacterium is Escherichia coli.
 19. The method according to claim 15,wherein the fungus is Saccharomyces, Schizosaccharomyces or Neurospora.20. The method according to claim 1, wherein the step (D) of selecting apurified nucleic acid having an intended functional property is carriedout in the following steps of: (i) transferring the purified nucleicacid into a third host cell to obtain a transformed cell; (ii) comparingthe property of the transformed cell with the property of a third hostcell that is not transformed; and (iii) determining whether or not thetransformed cell has an intended functional property, as a result of thecomparison.
 21. The method according to claim 20, wherein the step (D)of selecting a purified nucleic acid having an intended functionalproperty further includes the step of (iv) preparing a nucleic acidhaving an intended functional property from the selected cell.
 22. Themethod according to claim 20, wherein the third host cell is a mammaliancell.
 23. The method according to claim 20, wherein the third host cellis a human cell.
 24. The method according to claim 20, wherein the thirdhost cell is derived from the same species as the species from which thefirst host cell is derived.
 25. The method according to claim 1, whereinthe intended functional property is selected from the group consistingof induction of angiogenesis, tumor suppression, enhancement ofosteogenesis, induction of apoptosis, cytokine secretion, induction ofdendrites, suppression of arteriosclerosis, suppression of diabetes;suppression of autoimmune diseases; suppression of Alzheimer's disease,suppression of Parkinson's disease, protection of nerve cells andcombinations thereof
 26. A kit for isolating a nucleic acid having anintended functional property, comprising: (A) a nucleic acid transfervector to be transferred into a plurality of first host cells in orderto transform said first host cells; and (B) a second host cell forpreparing a purified nucleic acid from a cell selected from thetransformed first host cells.
 27. The kit according to claim 26, whereinthe nucleic acid transfer vector is a viral envelope, liposome orliposome containing at least one protein from a viral envelope.
 28. Thekit according to the claim 26, wherein the first host cells aremammalian cells.
 29. The kit according to claim 26, wherein the firsthost cells are human cells.
 30. The kit according to claim 26, whereinthe viral envelope is derived from wild-type or recombinant viruses. 31.The kit according to the 26, wherein the viral envelope is derived froma virus belonging to a family selected from the group consisting ofRetroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae,Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxyiridae,Herpesviridae, Baculoviridae and Hepadnaviridae.
 32. The kit accordingto claim 26, wherein the virus is derived from viruses belonging to thefamily Paramyxoviridae.
 33. The kit according to claim 26, wherein thevirus is HVJ.
 34. The kit according to claim 26, wherein the vector is aviral envelope vector.
 35. The kit according to claim 26, wherein thevector is a vector containing a protein prepared from a viral envelopeand a liposome.
 36. The kit according to claim 35, wherein the proteinprepared from viral envelope is a protein selected from the groupconsisting of F protein, HNprotein, NP protein and a combinationthereof.
 37. The kit according to claim 26, wherein the second host cellis a bacterium or fungus.
 38. The kit according to claim 26, furthercomprising a nucleic acid for preparing a nucleic acid to be transferredinto the first host cells.
 39. The kit according to claim 26, furthercomprising a reagent to be used for determining whether or not thepurified nucleic acid has an intended functional property.
 40. The kitaccording to claim 37, wherein the bacterium belongs to a genus selectedfrom the group consisting of Escherichia, Bacillus, Streptococcus,Staphylococcus, Haemophilus, Neisseria, Actinobacillus andAcinetobacter.
 41. The kit according to claim 40, wherein the bacteriumis Escherichia coli.
 42. The kit according to claim 37, wherein thefungus is Saccharomyces, Schizosaccharomyces or Neurospora.
 43. Anucleic acid isolated by the method according to claim
 1. 44. Use of aviral envelope for isolating a nucleic acid having an intendedfunctional property.
 45. Use of a liposome containing at least oneprotein from a viral envelope, for isolating a nucleic acid having anintended functional property.